Lipo-glycopeptide cleavable derivatives and uses thereof

ABSTRACT

The present invention provides certain lipo-glycopeptide cleavable derivatives and methods for using the same for the treatment of bacterial infections, for example, pulmonary bacterial infections. The LGPC derivatives include a cleavable moiety that in certain embodiments, is designed to allow for cellular uptake and/or a more rapid clearance of the glycopeptide metabolite (i.e., the cleaved glycopeptide) from the site of administration (e.g., the lung) as compared to the uncleaved LGPC.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 371 national phase entry of PCT Application No.PCT/US2018/033963, filed May 22, 2018, which claims priority from U.S.Provisional Application Ser. No. 62/509,378, filed May 22, 2017; U.S.Provisional Application Ser. No. 62/518,280, filed Jun. 12, 2017; andU.S. Provisional Application Ser. No. 62/560,413, filed Sep. 19, 2017,the disclosures of each of which is incorporated by reference herein intheir entireties.

BACKGROUND OF THE INVENTION

The high frequency of multidrug resistant bacteria, and in particular,Gram-positive bacteria, both in the hospital setting and the communitypresent a significant challenge for the management of infections (Krauseet al. (2008). Antimicrobial Agents and Chemotherapy 52(7), pp.2647-2652, incorporated by reference herein in its entirety for allpurposes).

The treatment of invasive Staphylococcus aureus (S. aureus) infectionshas relied significantly on vancomycin. However, the treatment andmanagement of such infections is a therapeutic challenge because certainS. aureus isolates, and in particular, methicillin-resistant S. aureusisolates, have been shown to be resistant to vancomycin (Shaw et al.(2005). Antimicrobial Agents and Chemotherapy 49(1), pp. 195-201; Mendeset al. (2015). Antimicrobial Agents and Chemotherapy 59(3), pp.1811-1814, each of which is incorporated by reference herein in itsentirety for all purposes).

Because of the resistance displayed by many Gram-positive organisms toantibiotics, and the general lack of susceptibility to existingantibiotics, there is a need for new therapeutic strategies to combatinfections due to these bacteria. The present invention addresses thisand other needs.

SUMMARY OF THE INVENTION

In one aspect, the present invention addresses the need for newantibiotics and treatment methods by providing certain glycopeptidescontaining a primary amino conjugated lipophilic moiety that iscleavable by enzymatic hydrolysis, and methods for using the same. Thelipophilic moiety is conjugated to the primary amino group via afunctional group that can undergo enzymatic hydrolysis. Glycopeptides ofthe present invention are referred to herein in various embodiments, aslipo-glycopeptide cleavable (LGPC) derivatives. Without being bound byany particular theory or mechanism, it is believed that the cleavage ofthe lipophilic moiety promotes clearance of the glycopeptide from thesite of administration. In one embodiment, the LGPC derivative clearsmore rapidly from the site of administration (e.g., the lung) ascompared to a structurally similar glycopeptide having a non-cleavablelipophilic moiety conjugated to the counterpart primary amino group.

In one embodiment of a LGPC derivative, a compound of Formula (I), or apharmaceutically acceptable salt thereof, is provided:Glycopeptide-R¹  (I)wherein,

R¹ is conjugated to the Glycopeptide at a primary amine group of theGlycopeptide;

R¹ is —(CH₂)_(n1)—C(O)—O—(CH₂)_(n2)—CH₃;—(CH₂)_(n1)—C(O)—NH—(CH₂)_(n2)—CH₃; —C(O)—(CH₂)_(n2)—CH₃;—(CH₂)_(n1)—NH—C(O)—(CH₂)_(n2)—CH₃; —(CH₂)_(n1)—O—C(O)—(CH₂)_(n2)—CH₃;—(CH₂)_(n1)—O—C(O)—NH—(CH₂)_(n2)—CH₃;—(CH₂)_(n1)—O—(CO)—O—(CH₂)_(n2)—CH₃ or—(CH₂)_(n1)—NH—C(O)—O—(CH₂)_(n2)—CH₃;

n1 is 1, 2, 3, 4 or 5; and

n2 is 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

In one embodiment of a compound of Formula (I), or a pharmaceuticallyacceptable salt thereof, the Glycopeptide is vancomycin, telavancin,chloroeremomycin or decaplanin. In a further embodiment, theGlycopeptide is telavancin, chloroeremomycin or decaplanin.

In one embodiment of a compound of Formula (I), or a pharmaceuticallyacceptable salt thereof, R₁ is —(CH₂)_(n1)—C(O)—NH—(CH₂)_(n2)—CH₃;—(CH₂)_(n1)—NH—C(O)—(CH₂)_(n2)—CH₃ or —(CH₂)_(n1)—O—C(O)—(CH₂)_(n2)—CH₃;n1 is 1 or 2, and n2 is 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In afurther embodiment, the Glycopeptide is vancomycin. In an even furtherembodiment, n2 is 6, 7, 8, 9, 10, 11, 12, or 14.

In one embodiment of a compound of Formula (I), or a pharmaceuticallyacceptable salt thereof, R₁ is —(CH₂)_(n1)—NH—C(O)—(CH₂)_(n2)—CH₃ or—(CH₂)_(n1)—O—C(O)—(CH₂)_(n2)—CH₃; n1 is 1, 2, 3 or 4, and n2 is 9, 10or 11. In even a further embodiment, the Glycopeptide is vancomycin.

In one embodiment of a compound of Formula (I), or a pharmaceuticallyacceptable salt thereof, R₁ is —(CH₂)_(n1)—NH—C(O)—(CH₂)_(n2)—CH₃; n1 is1, 2, 3 or 4, n2 is 9, 10 or 11. In even a further embodiment, theGlycopeptide is vancomycin.

In one embodiment of a compound of Formula (I), or a pharmaceuticallyacceptable salt thereof, R₁ is —(CH₂)_(n1)—O—C(O)—(CH₂)_(n2)—CH₃; n1 is1, 2, 3 or 4, and n2 is 9, 10 or 11. In a further embodiment, theGlycopeptide is vancomycin. In a further embodiment, n1 is 2 and n2 is10.

In one embodiment, a compound of the disclosure is represented byFormula (II), or a pharmaceutically acceptable salt thereof:

wherein,

R¹ is —(CH₂)_(n1)—C(O)—O—(CH₂)_(n2)—CH₃;—(CH₂)_(n1)—C(O)—NH—(CH₂)_(n2)—CH₃; —C(O)—(CH₂)_(n2)—CH₃;—(CH₂)_(n1)—NH—C(O)—(CH₂)_(n2)—CH₃; —(CH₂)_(n1)—O—C(O)—(CH₂)_(n2)—CH₃;—(CH₂)_(n1)—O—C(O)—NH—(CH₂)_(n2)—CH₃;—(CH₂)_(n1)—O—(CO)—O—(CH₂)_(n2)—CH₃ or—(CH₂)_(n1)—NH—C(O)—O—(CH₂)_(n2)—CH₃;

n1 is 1, 2, 3, 4 or 5;

n2 is 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

R² is OH or NH—(CH₂)_(q)—R⁵;

q is 1, 2, 3, 4, or 5;

R³ is

R⁴ is H or CH₂—NH—CH₂—PO₃H₂; and

R⁵ is —N(CH₃)₂, —N⁺(CH₃)₃, —N⁺(CH₃)₂(n-C₁₄H₂₉), or

In one embodiment of a compound of Formula (II), or a pharmaceuticallyacceptable salt thereof, R₁ is —(CH₂)_(n1)—C(O)—NH—(CH₂)_(n2)—CH₃;—(CH₂)_(n1)—NH—C(O)—(CH₂)_(n2)—CH₃ or —(CH₂)_(n1)—O—C(O)—(CH₂)_(n2)—CH₃;n1 is 1 or 2, and n2 is 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In afurther embodiment, R² is OH or NH—(CH₂)₃—N(CH₃)₂.; and R³ is H. In afurther embodiment, n2 is 6, 7, 8, 9, 10, 11, 12, or 14.

In one embodiment of a compound of Formula (II), or a pharmaceuticallyacceptable salt thereof, R₁ is —(CH₂)_(n1)—NH—C(O)—(CH₂)_(n2)—CH₃ or—(CH₂)_(n1)—O—C(O)—(CH₂)_(n2)—CH₃. In a further embodiment, n1 is 1, 2,3 or 4, and n2 is 9, 10 or 11. In even a further embodiment, n1 is 2 andn2 is 10. In still even a further embodiment, R² is OH, R³ is H and R⁴is H.

In one embodiment of a compound of Formula (II), or a pharmaceuticallyacceptable salt thereof, R₁ is —(CH₂)_(n1)—NH—C(O)—(CH₂)_(n2)—CH₃. In afurther embodiment, n1 is 1, 2, 3 or 4, and n2 is 9, 10 or 11. In even afurther embodiment, n1 is 2 and n2 is 10. In still even a furtherembodiment, R² is OH, R³ is H and R⁴ is H.

In one embodiment of a compound of Formula (II), or a pharmaceuticallyacceptable salt thereof, R₁ is —(CH₂)_(n1)—O—C(O)—(CH₂)_(n2)—CH₃. In afurther embodiment, n1 is 1, 2, 3 or 4, and n2 is 9, 10 or 11. In even afurther embodiment, n1 is 2 and n2 is 10. In still even a furtherembodiment, R² is OH, R³ is H and R⁴ is H.

In one embodiment of a compound of Formula (II), or a pharmaceuticallyacceptable salt thereof, R₁ is —(CH₂)_(n1)—C(O)O—(CH₂)_(n2)—CH₃ or—(CH₂)_(n1)—C(O)NH—(CH₂)_(n2)—CH₃. In a further embodiment, n1 is 1, 2,3 or 4, and n2 is 9, 10 or 11. In even a further embodiment, n1 is 2 andn2 is 10. In still even a further embodiment, R² is OH, R³ is H and R⁴is H.

In one embodiment of a compound of Formula (II), or a pharmaceuticallyacceptable salt thereof, R₁ is —C(O)—(CH₂)_(n2)—CH₃. In a furtherembodiment, n2 is 9, 10 or 11. In even a further embodiment, n1 is 2 andn2 is 10. In even a further embodiment, R² is OH, R³ is H and R⁴ is H.

In another embodiment of a compound of Formula (II), or apharmaceutically acceptable salt thereof, R₂ is —NH—(CH₂)_(q)—R³. In afurther embodiment, q is 3 and R₃ is —N(CH₃)₂. In even a furtherembodiment, n1 is 1, 2, 3 or 4 and n2 is 9, 10 or 11. In still even afurther embodiment, R³ is H and R⁴ is H. In even a further embodiment,R¹ includes an amide group.

In another embodiment of a compound of Formula (II), or apharmaceutically acceptable salt thereof, R₂ is —NH—(CH₂)_(q)—R³. In afurther embodiment, q is 3 and R₃ is —N(CH₃)₂. In even a furtherembodiment, n1 is 1, 2, 3 or 4 and n2 is 9, 10 or 11. In still even afurther embodiment, R³ is H and R⁴ is H. In even a further embodiment,R¹ includes an ester group. In another embodiment of a compound ofFormula (II), or a pharmaceutically acceptable salt thereof, n1 is 1, 2,3 or 4, and n2 is 9, 10 or 11. In even a further embodiment, n1 is 2 andn2 is 10. In still even a further embodiment, R² is OH, R³ is

and R⁴ is H.

In one embodiment of a compound of Formula (II), or a pharmaceuticallyacceptable salt thereof, n1 is 1, 2, 3 or 4, n2 is 9, 10 or 11, and R⁴is CH₂—NH—CH₂—PO₃H₂. In a further embodiment, n1 is 2 and n2 is 10. Ineven a further embodiment, R² is OH and R³ is H.

In yet another aspect of the invention, a composition is providedcomprising an effective amount of a compound of Formula (I), Formula(II), or a pharmaceutically acceptable salt of one of the foregoing. Ina further embodiment, the composition is a dry powder.

In one embodiment, the composition provided herein comprises a pluralityof nanoparticles of the compound of Formula (I) or (II), or apharmaceutically acceptable salt thereof, in association with a polymer.In further embodiments, the compositions are suitable for administrationvia the pulmonary route, e.g., via inhalation with a nebulizer, a drypowder inhaler or a metered dose inhaler.

In yet another aspect of the invention, a method is provided fortreating a bacterial infection in a patient in need thereof. Thebacterial infection can comprise planktonic bacteria and/or bacteriapresent in a biofilm. The method comprises administering to the patientin need of treatment, a composition comprising a therapeuticallyeffective amount of a compound of Formula (I), Formula (II), or apharmaceutically acceptable salt of a compound of Formula (I) or (II).In one embodiment, the bacterial infection is a gram positive bacterialinfection. In a further embodiment, the bacterial infection is apulmonary bacterial infection. As such, in one embodiment, theadministering is via the pulmonary route, e.g., via dry powder inhaler.

In another embodiment, the administering is via the intravenous (IV)route for the treatment of a localized bacterial infection. In oneembodiment, the compound administered to the patient is a compound ofFormula (II) wherein n1 is 2, 3 or 4, and n2 is 9, 10 or 11. In afurther embodiment, R¹ includes an ester moiety. In even a furtherembodiment, n1 is 2 and n2 is 10. In still even a further embodiment, R²is OH, R³ is H and R⁴ is H.

In one embodiment, the bacterial infection is an infection caused by aGram-positive microorganism. In one embodiment, the bacterial infectionis a pulmonary bacterial infection. In a further embodiment, thepulmonary bacterial infection is a Gram-positive cocci infection. Ineven a further embodiment, the pulmonary bacterial infection is aStaphylococcus, Enterococcus or Streptococcus infection. In even afurther embodiment, the administering comprises administering viainhalation.

Streptococcus pneumoniae is treated, in one embodiment, in a patientthat has been diagnosed with community-acquired pneumonia,hospital-acquired pneumonia or purulent meningitis. An Enterococcusinfection is treated, in one embodiment, in a patient that has beendiagnosed with a urinary-catheter related infection. A Staphylococcusinfection, e.g., S. aureus is treated in one embodiment, in a patientthat has been diagnosed with mechanical ventilation-associatedpneumonia.

In one embodiment of the present methods, a Staphylococcus infection istreated and is a Staphylococcus aureus (S. aureus) infection. In anotherembodiment, the S. aureus infection is a methicillin-resistant S. aureus(MRSA) infection.

In one embodiment of the present methods, an Enterococcus infection istreated and is an Enterococcus faecalis (E. faecalis) infection. Inanother embodiment of the present methods, the Enterococcus infection isan Enterococcus faecium (E. faecium) infection.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the reductive amination of vancomycin to arrive at a LGPCderivative. The reaction occurs at the primary amine of vancomycin.

FIG. 2 shows one reaction scheme for aldehyde preparation.

FIG. 3 is a graph of the percent degradation of RV62 and RV65 as afunction of time, as determined by HPLC.

FIG. 4 is a graph of the percent degradation of certain LGPC derivatives(RV65, RV88, RV89, RV90), after incubation with esterase, as determinedby HPLC.

FIG. 5 is a graph showing the hydrolysis of RV62 and RV65 as a functionof time, after incubation in rat plasma over 24 h.

FIG. 6 is a graph showing the levels of RV62 (μg/g) and RV82 (μg/g) inthe lung as a function of time.

FIG. 7 is a graph showing the levels of RV62 (μg/g) and RV82 (μg/g) inblood plasma as a function of time.

DETAILED DESCRIPTION OF THE INVENTION

The high frequency of multidrug resistant bacteria, and in particular,Gram-positive bacteria, both in the healthcare setting and the communitypresent a significant challenge for the management of infections (Krauseet al. (2008). Antimicrobial Agents and Chemotherapy 52(7), pp.2647-2652, incorporated by reference herein in its entirety for allpurposes). Moreover, methicillin resistant S. aureus (MRSA) infectionsin cystic fibrosis (CF) patients is a concern, and there is a lack ofclinical data regarding approaches to eradicate such infections (Gossand Muhlebach (2011). Journal of Cystic Fibrosis 10, pp. 298-306,incorporated by reference herein in its entirety for all purposes).

Due to the high frequency of resistant pathogens, novel compounds andmethods are needed to treat infections due to such pathogens. Moreover,it has been found that semi synthetic glycopeptides containing primaryamino conjugated lipophilic moieties can accumulate in tissue and canexhibit long half-lives at the site of administration followingadministration (e.g., administration via inhalation). As such,glycopeptides that promote clearance from the site of administration areneeded.

The present invention addresses the need for new antibiotics andtreatment methods by providing certain glycopeptides containing aprimary amino conjugated lipophilic moiety that is cleavable byenzymatic hydrolysis, and methods for using the same. The lipophilicmoiety is conjugated to the primary amino group via a functional groupthat is capable of undergoing enzymatic hydrolysis. The functional groupthat undergoes enzymatic hydrolysis, in one embodiment, in conjugated tothe primary amino group via a straight chain or branched alkyl group,e.g., a methyl, ethyl, propyl or butyl group. In another embodiment, thefunctional group is an amide that comprises the nitrogen atom from theprimary amino group of the glycopeptide.

Glycopeptides of the present invention are referred to herein in variousembodiments, as lipo-glycopeptide cleavable (LGPC) derivatives. Withoutbeing bound by any particular theory or mechanism, it is believed thatthe cleavage of the lipophilic moiety promotes clearance of theglycopeptide from the site of administration. In one embodiment, theLGPC derivative clears more rapidly from the site of administration(e.g., the lung) as compared to a structurally similar glycopeptidehaving a non-cleavable lipophilic moiety conjugated to the counterpartprimary amino group.

As an example, in one embodiment, a glycopeptide containing a cleavablelipophilic group attached to a primary amino group of the glycopeptideclears from the site of administration at a faster rate than aglycopeptide having a non-cleavable lipophilic group attached to thesame primary amino group. In another embodiment, the LGPC has ahalf-life (T_(1/2)) at the site of administration that is shorter thanthe T_(1/2) of a glycopeptide having a non-cleavable lipophilic groupattached to the primary amino group. The comparison of clearance is madein one embodiment, between glycopeptides having the same core structure,but for the different primary amino group conjugated moieties. Oneembodiment of an appropriate comparison is shown in Table A.

TABLE A LGPC derivative primary amino Semi-synthetic glycopeptideprimary conjugated cleavable moiety amino conjugated non-cleavablemoiety -(alkyl)_(n1)-Y¹-lipophilic group vs. -(alkyl)_(n1)-Y²-lipophilicgroup Each n1 is the same for each comparison, or differs by 1, 2 or 3carbon atoms. Y¹ is a functional group that can undergo enzymatichydrolysis, e.g., —O—C(O)—; —NH—C(O)—; —C(O)—O—; —C(O)—NH—; —O—C(O)—NH;NH—C(O)—O; O—C(O)—O Y² is a functional group that cannot undergoenzymatic hydrolysis, e.g., —O—; —NH—; —S—S—; —SO₂—; Alkyl is eithersubstituted or unsubstituted. Each lipophilic group is the same, ordiffers in length by one carbon or two carbon atoms. The lipophilicgroup, in one embodiment, is an alkyl group, and can be straight chainor branched. In a further embodiment, the alkyl group is substituted atone, two or three carbon atoms.

As such, in one embodiment, the LGPCs provided herein are intended topromote glycopeptide clearance from tissue, for example, increasedclearance from the lung after local administration via inhalation. Ascleavage of the delivered LGPC derivative occurs over a time T₁, aneffective amount of LGPC derivative can remain at the site of actionduring T₁, or a portion thereof.

Cleavage, in one embodiment, is via an esterase. In another embodiment,cleavage occurs in vivo via an amidase. In another embodiment, cleavageoccurs in vivo via a protease such as a peptidase.

Importantly, the compounds provided herein would not be consideredprodrugs, even though they each contain a labile moiety. Rather, theuncleaved LGPCs provided herein are more active than their cleavedmetabolite.

In one embodiment, the LGPC derivative provided herein has a shorterT_(1/2) than a counterpart uncleavable lipophilic derivatizedglycopeptide. In one embodiment, the T_(1/2) of the LGPC is about 5-75%of the T_(1/2) of the uncleavable lipophilic derivatized glycopeptide,including about 5-10%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%,5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 10-15%, 10-20%, 10-25%, 10-30%,10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%,15-20%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%,15-65%, 15-70%, 15-75%, 20-25%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%,20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 25-30%, 25-35%, 25-40%, 25-45%,25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 30-35%, 30-40%, 30-45%,30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 35-40%, 35-45%, 35-50%,35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 40-45%, 40-50%, 40-55%, 40-60%,40-65%, 40-70%, 40-75%, 45-50%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%,50-55%, 50-60%, 50-65%, 50-70%, 50-75%, 55-60%, 55-65%, 55-70%, 55-75%,60-65%, 60-70%, 60-75%, 65-70%, 65-75%, or 70-75% of the T_(1/2) of theuncleavable lipophilic derivatized glycopeptide.

In one embodiment, the LGPC derivative provided herein has a fasterclearance rate from the site of administration than a counterpartuncleavable lipophilic derivatized glycopeptide. In one embodiment, theclearance rate of the LGPC is about 5-75% of the clearance rate of theuncleavable lipophilic derivatized glycopeptide, including about 5-10%,5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%,5-65%, 5-70%, 10-15%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%,10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 15-20%, 15-25%, 15-30%,15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%,20-25%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%,20-70%, 20-75%, 25-30%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%,25-65%, 25-70%, 25-75%, 30-35%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%,30-65%, 30-70%, 30-75%, 35-40%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%,35-70%, 35-75%, 40-45%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%,45-50%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 50-55%, 50-60%, 50-65%,50-70%, 50-75%, 55-60%, 55-65%, 55-70%, 55-75%, 60-65%, 60-70%, 60-75%,65-70%, 65-75%, or 70-75% of the clearance rate of the uncleavablelipophilic derivatized glycopeptide.

In one embodiment, the LGPC derivative provided herein has a minimuminhibitory concentration (MIC) against a particular bacterium that islower than its cleaved metabolite. In particular embodiments the MIC ofthe LGPC is about 5-75% of the MIC of the cleaved metabolite, includingabout 5-10%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%,5-55%, 5-60%, 5-65%, 5-70%, 10-15%, 10-20%, 10-25%, 10-30%, 10-35%,10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 15-20%,15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%,15-70%, 15-75%, 20-25%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%,20-60%, 20-65%, 20-70%, 20-75%, 25-30%, 25-35%, 25-40%, 25-45%, 25-50%,25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 30-35%, 30-40%, 30-45%, 30-50%,30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 35-40%, 35-45%, 35-50%, 35-55%,35-60%, 35-65%, 35-70%, 35-75%, 40-45%, 40-50%, 40-55%, 40-60%, 40-65%,40-70%, 40-75%, 45-50%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 50-55%,50-60%, 50-65%, 50-70%, 50-75%, 55-60%, 55-65%, 55-70%, 55-75%, 60-65%,60-70%, 60-75%, 65-70%, 65-75%, or 70-75% of the MIC of the cleavedglycopeptide. In certain embodiments, the bacterium is a Gram-positivebacterium. In a further embodiment, the bacterium ismethicillin-resistant Staphylococcus aureus (MRSA).

In the methods provided herein, the bacterial infection can compriseplanktonic bacteria, bacterial biofilm, or a combination thereof.

One or more compounds provided herein, e.g., a LGPC of Formula (I) or(II), or a pharmaceutically acceptable salt thereof, is delivered to apatient in need of treatment of the bacterial infection. In oneembodiment, the bacterial infection is a pulmonary bacterial infectionand the composition is administered via the pulmonary route (e.g.,inhalation).

“Pharmaceutically acceptable salt” includes both acid and base additionsalts. A pharmaceutically acceptable addition salt refers to those saltswhich retain the biological effectiveness and properties of the freebases, which are not biologically or otherwise undesirable, and whichare formed with inorganic acids such as, but are not limited to,hydrochloric acid (HCl), hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as, but not limitedto, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid,ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid,4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid,capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid,citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonicacid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid,fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid,gluconic acid, glucuronic acid, glutamic acid, glutaric acid,2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuricacid, isobutyric acid, lactic acid (e.g., as lactate), lactobionic acid,lauric acid, maleic acid, malic acid, malonic acid, mandelic acid,methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid,naphthalene-2-sulfonic acid, l-hydroxy-2-naphthoic acid, nicotinic acid,oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid,propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid,4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, aceticacid (e.g., as acetate), tartaric acid, thiocyanic acid,p-toluenesulfonic acid, trifluoroacetic acid (TFA), undecylenic acid,and the like. In one embodiment, the pharmaceutically acceptable salt isHCl, TFA, lactate or acetate.

A pharmaceutically acceptable base addition salt retains the biologicaleffectiveness and properties of the free acids, which are notbiologically or otherwise undesirable. These salts are prepared fromaddition of an inorganic base or an organic base to the free acid. Saltsderived from inorganic bases include, but are not limited to, thesodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc,copper, manganese, aluminum salts and the like. Inorganic salts includethe ammonium, sodium, potassium, calcium, and magnesium salts. Saltsderived from organic bases include, but are not limited to, salts ofprimary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as ammonia, isopropylamine, trimethylamine,diethylamine, triethylamine, tripropylamine, diethanolamine,ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol,dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine,glucosamine, methylglucamine, theobromine, triethanolamine,tromethamine, purines, piperazine, piperidine, A-ethylpiperidine,polyamine resins and the like. Organic bases that can be used to form apharmaceutically acceptable salt include isopropylamine, diethylamine,ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

In one aspect, the present invention relates to methods for treatingbacterial infections, for example, Gram-positive bacterial infections,and diseases associated with the same. In one embodiment, theGram-positive bacterial infection is a pulmonary infection. In oneembodiment, the infection is a bacterial biofilm infection. The method,in one embodiment, comprises administering to a patient in need thereof,a composition comprising an effective amount of a compound of Formula(I) or Formula (II), or a pharmaceutically acceptable salt thereof. Thecomposition can be administered by any route. In the case of a pulmonaryinfection, in one embodiment, the composition is administered via anebulizer, dry powder inhaler or a metered dose inhaler.

In one aspect of the present invention, an LGPC derivative of Formula(I) or (II), or a pharmaceutically acceptable salt, is provided. TheLGPC derivatives of the present invention include a biologically-labilemoiety (e.g., amide, ester) that is conjugated to a glycopeptide via anamine group, e.g., a primary amine, on the glycopeptide. Uponadministration, the biologically-labile moiety undergoes cleavage (e.g.,via hydrolysis or enzymatic cleavage), providing one or moreglycopeptide metabolites. In some embodiments, the metabolite provides adecreased residence time in the lungs compared to the unmetabolizedcompounds, thereby assisting in elimination of the therapeutic agentfrom the organ (e.g., lung in the case of pulmonary administration).

The compounds and formula described herein are set forth graphicallywithout depicting stereochemistry. However, one of ordinary skill in theart will understand that the LGPC derivatives described herein each havea stereochemical configuration. In some embodiments, a stereoisomer(e.g., enantiomer, diastereomer) or a combination of stereoisomers ofthe respective LGPC derivative are provided.

In one embodiment, the present invention is directed to a compound ofFormula (I), or a pharmaceutically acceptable salt thereof:Glycopeptide-R¹  (I)

R¹ is conjugated to the Glycopeptide at a primary amine group of theGlycopeptide;

R¹ is —(CH₂)_(n1)—C(O)—O—(CH₂)_(n2)—CH₃;—(CH₂)_(n1)—C(O)—NH—(CH₂)_(n2)—CH₃; —C(O)—(CH₂)_(n2)—CH₃;—(CH₂)_(n1)—NH—C(O)—(CH₂)_(n2)—CH₃; —(CH₂)_(n1)—O—C(O)—(CH₂)_(n2)—CH₃;—(CH₂)_(n1)—O—C(O)—NH—(CH₂)_(n2)—CH₃;—(CH₂)_(n1)—O—(CO)—O—(CH₂)_(n2)—CH₃ or—(CH₂)_(n1)—NH—C(O)—O—(CH₂)_(n2)—CH₃;

n1 is 1, 2, 3, 4 or 5; and

n2 is 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

In one embodiment of a compound of Formula (I), or a pharmaceuticallyacceptable salt thereof, the Glycopeptide is vancomycin, telavancin,chloroeremomycin or decaplanin. In a further embodiment, theGlycopeptide is telavancin, chloroeremomycin or decaplanin.

The structures of hundreds of natural and semisynthetic glycopeptideshave been determined. These structures are highly related and fallwithin five structural subtypes, I-V, and the present invention is notlimited to a particular subtype, so long as the glycopeptide includes aprimary amine group to conjugate the R¹ group. Of the varying structuralsubtypes, type I structures contain aliphatic chains, whereas types II,III, and IV include aromatic side chains within these amino acids.Unlike types I and II, types III and IV contain an extra F-O-G ringsystem. Type IV compounds have, in addition, a long fatty-acid chainattached to the sugar moiety. Structures of type V, such ascomplestatin, chloropeptin I, and kistamincin A and B, contain thecharacteristic tryptophan moiety linked to the central amino acid.

In one embodiment, one of the glycopeptides described in PCT publicationno. WO 2014/085526, the disclosure of which is incorporated by referenceherein for all purposes, can be used as the glycopeptide set forth inFormula (I).

In one embodiment of Formula (I), the Glycopeptide is A477, A35512,A40926, A41030, A42867, A47934, A80407, A82846, A83850, A84575, AB-65,actaplanin, actinoidin, ardacin, avoparcin, azureomycin,chloroorienticin chloropolysporin, chloroeremomycin, decaplanin,N-demethylvancomycin, eremomycin, galacardin, helvecardin A, helvecardinB, izupeptin, kibdelin, LL-AM374, mannopeptin, MM45289, MM47761,MM47766. MM55266, MM55270, OA-7653, orienticin, parvodicin, ristocetin,ristomycin, synmonicin, teicoplanin, telavancin, UK-68597, UK-69542,UK-72051, vancomycin, or a pharmaceutically acceptable salt of one ofthe foregoing.

In one embodiment of Formula (I), the Glycopeptide is vancomycin. In oneembodiment of Formula (I), the Glycopeptide is telavancin. In oneembodiment of Formula (I), the Glycopeptide is chloroeremomycin. In oneembodiment of Formula (I), the Glycopeptide is decaplanin.

In one embodiment of a compound of Formula (I), or a pharmaceuticallyacceptable salt thereof, n1 is 2 or 3; and n2 is 8, 9, 10, 11 or 12. Ineven a further embodiment, n1 is 2 and n2 is 10. In a furtherembodiment, the Glycopeptide is vancomycin, telavancin orchloroeremomycin. In even a further embodiment, the Glycopeptide isvancomycin.

In one embodiment of a compound of Formula (I), or a pharmaceuticallyacceptable salt thereof, R¹ is —(CH₂)_(n1)—C(O)—O—(CH₂)_(n2)—CH₃. In afurther embodiment, n1 is 1, 2 or 3; and n2 is 8, 9, 10, 11 or 12. Ineven a further embodiment, n1 is 2 and n2 is 10. In a furtherembodiment, the Glycopeptide is vancomycin, telavancin orchloroeremomycin. In even a further embodiment, the Glycopeptide isvancomycin.

In one embodiment of a compound of Formula (I), or a pharmaceuticallyacceptable salt thereof, R¹ is —(CH₂)_(n1)—C(O)—NH—(CH₂)_(n2)—CH₃. In afurther embodiment, n1 is 2 or 3; and n2 is 8, 9, 10, 11 or 12. In evena further embodiment, n1 is 2 and n2 is 10. In a further embodiment, theGlycopeptide is vancomycin, telavancin or chloroeremomycin. In even afurther embodiment, the Glycopeptide is vancomycin.

In one embodiment of a compound of Formula (I), or a pharmaceuticallyacceptable salt thereof, R¹ is —(CH₂)_(n1)—NH—C(O)—(CH₂)_(n2)—CH₃. In afurther embodiment, n1 is 1, 2 or 3; and n2 is 8, 9, 10, 11 or 12. Ineven a further embodiment, n1 is 2 and n2 is 10. In a furtherembodiment, the Glycopeptide is vancomycin, telavancin orchloroeremomycin. In even a further embodiment, the Glycopeptide isvancomycin.

In one embodiment of a compound of Formula (I), or a pharmaceuticallyacceptable salt thereof, R¹ is —(CH₂)_(n1)—O—C(O)—(CH₂)_(n2)—CH₃. In afurther embodiment, n1 is 2 or 3; and n2 is 8, 9, 10, 11 or 12. In evena further embodiment, n1 is 2 and n2 is 10. In a further embodiment, theGlycopeptide is vancomycin, telavancin or chloroeremomycin. In even afurther embodiment, the Glycopeptide is vancomycin

In one embodiment of a compound of Formula (I), or a pharmaceuticallyacceptable salt thereof, R¹ is —C(O)—(CH₂)_(n2)—CH₃. In a furtherembodiment, n2 is 8, 9, 10, 11 or 12. In even a further embodiment, n2is 10. In a further embodiment, the Glycopeptide is vancomycin,telavancin or chloroeremomycin. In even a further embodiment, theGlycopeptide is vancomycin.

In one embodiment of a compound of Formula (I), or a pharmaceuticallyacceptable salt thereof, n1 is 1, 2 or 3; and n2 is 10, 11, 12 or 13. Ineven a further embodiment, n1 is 2 and n2 is 10 or 11. In a furtherembodiment, the Glycopeptide is vancomycin, telavancin orchloroeremomycin. In even a further embodiment, the Glycopeptide isvancomycin.

In one embodiment of a compound of Formula (I), or a pharmaceuticallyacceptable salt thereof, R¹ is —(CH₂)_(n1)—C(O)—O—(CH₂)_(n2)—CH₃. In afurther embodiment, n1 is 1, 2 or 3; and n2 is 10, 11, 12 or 13. In evena further embodiment, n1 is 2 and n2 is 10 or 11. a further embodiment,the Glycopeptide is vancomycin, telavancin or chloroeremomycin. In evena further embodiment, the Glycopeptide is vancomycin.

In one embodiment of a compound of Formula (I), or a pharmaceuticallyacceptable salt thereof, R¹ is —(CH₂)_(n1)—C(O)—NH—(CH₂)_(n2)—CH₃. In afurther embodiment, n1 is 2 or 3; and n2 is 10, 11, 12 or 13. In even afurther embodiment, n1 is 1, 2 or 3 and n2 is 10 or 11. In a furtherembodiment, the Glycopeptide is vancomycin, telavancin orchloroeremomycin. In even a further embodiment, the Glycopeptide isvancomycin.

In one embodiment of a compound of Formula (I), or a pharmaceuticallyacceptable salt thereof, R¹ is —(CH₂)_(n1)—NH—C(O)—(CH₂)_(n2)—CH₃. In afurther embodiment, n1 is 1, 2 or 3; and n2 is 10, 11, 12 or 13. In evena further embodiment, n1 is 2 and n2 is 10 or 11. In a furtherembodiment, the Glycopeptide is vancomycin, telavancin orchloroeremomycin. In even a further embodiment, the Glycopeptide isvancomycin.

In one embodiment of a compound of Formula (I), or a pharmaceuticallyacceptable salt thereof, R¹ is —(CH₂)_(n1)—O—C(O)—(CH₂)_(n2)—CH₃. In afurther embodiment, n1 is 1, 2 or 3; and n2 is 10, 11, 12 or 13. In evena further embodiment, n1 is 2 and n2 is 10 or 11. In a furtherembodiment, the Glycopeptide is vancomycin, telavancin orchloroeremomycin. In even a further embodiment, the Glycopeptide isvancomycin

In one embodiment of a compound of Formula (I), or a pharmaceuticallyacceptable salt thereof, R¹ is —C(O)—(CH₂)_(n2)—CH₃. In a furtherembodiment, n2 is 10, 11, 12 or 13. In even a further embodiment, n2 is10 or 11. In a further embodiment, the Glycopeptide is vancomycin,telavancin or chloroeremomycin. In even a further embodiment, theGlycopeptide is vancomycin.

In another embodiment, a compound of Formula (II), or a pharmaceuticallyacceptable salt thereof, is provided:

wherein,

R¹ is —(CH₂)_(n1)—C(O)—O—(CH₂)_(n2)—CH₃;—(CH₂)_(n1)—C(O)—NH—(CH₂)_(n2)—CH₃; —C(O)—(CH₂)_(n2)—CH₃;—(CH₂)_(n1)—NH—C(O)—(CH₂)_(n2)—CH₃; —(CH₂)_(n1)—O—C(O)—(CH₂)_(n2)—CH₃;—(CH₂)_(n1)—O—C(O)—NH—(CH₂)_(n2)—CH₃;—(CH₂)_(n1)—O—(CO)—O—(CH₂)_(n2)—CH₃ or—(CH₂)_(n1)—NH—C(O)—O—(CH₂)_(n2)—CH₃;

n1 is 1, 2, 3, 4 or 5;

n2 is 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;

R² is OH or NH—(CH₂)_(q)—R⁵;

q is 1, 2, 3, 4, or 5;

R³ is H or

R⁴ is H or CH₂—NH—CH₂—PO₃H₂; and

R⁵ is —N(CH₃)₂, —N⁺(CH₃)₃, —N⁺(CH₃)₂(n-C₁₄H₂₉), or

In some embodiments of Formula (II), R² is OH. In a further embodiment,R⁴ is H.

In some embodiments of Formula (II), R² is OH. In a further embodiment,R⁴ is CH₂—NH—CH₂—PO₃H₂.

In one embodiment of a compound of Formula (II), or a pharmaceuticallyacceptable salt thereof, R² is —NH—(CH₂)₃—R³. In a further embodiment,R³ and R⁴ are H.

In one embodiment of a compound of Formula (II), or a pharmaceuticallyacceptable salt thereof, R² is —NH—(CH₂)₃—R³. In a further embodiment,R⁴ is CH₂—NH—CH₂—PO₃H₂.

In one embodiment of a compound of Formula (II), or a pharmaceuticallyacceptable salt thereof, R² is —NH—(CH₂)_(q)—R⁵. In a furtherembodiment, R² is —NH—(CH₂)₃—N(CH₃)₂. In another embodiment, R² is—NH—(CH₂)₃—N⁺(CH₃)₃. In yet other embodiments, R² is—NH—(CH₂)₃—N⁺(CH₃)₂(n-C₁₄H₂₉). In a further embodiment, R² is

In one embodiment of a compound of Formula (II), or a pharmaceuticallyacceptable salt thereof, R² is —NH—(CH₂)_(q)—N(CH₃)₂. In anotherembodiment, R² is —NH—(CH₂)_(q)—N⁺(CH₃)₃. In another embodiment, R² is—NH—(CH₂)_(q)—R⁵ and R⁵ is —N⁺(CH₃)₂(n-C₁₄H₂₉). In yet anotherembodiment, R² is —NH—(CH₂)_(q)—R⁵ and R⁵

In one embodiment of a compound of Formula (II), or a pharmaceuticallyacceptable salt thereof, R¹ is —(CH₂)_(n1)—O—C(O)—(CH₂)_(n2)—CH₃ or—(CH₂)_(n1)—NH—C(O)—(CH₂)_(n2)—CH₃. In a further embodiment, R² is OH,R³ is H and R⁴ is H. In even a further embodiment, n1 is 1, 2 or 3, n2is 9, 10, 11, 12, 13 or 14. In even a further embodiment, n1 is 2 and n2is 10. In yet even a further embodiment, R¹ is—(CH₂)_(n1)—O—C(O)—(CH₂)_(n2)—CH₃.

In one embodiment of a compound of Formula (II), or a pharmaceuticallyacceptable salt thereof, R¹ is —(CH₂)_(n1)—NH—C(O)—(CH₂)_(n2)—CH₃. In afurther embodiment, R² is OH, R³ is H and R⁴ is H. In even a furtherembodiment, n1 is 1, 2 or 3, n2 is 9, 10, 11, 12, 13 or 14. In even afurther embodiment, n1 is 2 and n2 is 10.

In one embodiment of a compound of Formula (II), or a pharmaceuticallyacceptable salt thereof, R¹ is —(CH₂)_(n1)—O—C(O)—(CH₂)_(n2)—CH₃. In afurther embodiment, R² is OH, R³ is H and R⁴ is H. In even a furtherembodiment, n1 is 1, 2 or 3, n2 is 9, 10, 11, 12, 13 or 14. In even afurther embodiment, n1 is 2 and n2 is 10.

In one embodiment of a compound of Formula (II), or a pharmaceuticallyacceptable salt thereof, R¹ is —(CH₂)_(n1)—C(O)—O—(CH₂)_(n2)—CH₃. In afurther embodiment, R² is OH, R³ is H and R⁴ is H. In even a furtherembodiment, n1 is 1, 2 or 3, n2 is 9, 10, 11, 12, 13 or 14. In even afurther embodiment, n1 is 2 and n2 is 10.

In one embodiment of a compound of Formula (II), or a pharmaceuticallyacceptable salt thereof, R¹ is —(CH₂)_(n1)—C(O)—NH—(CH₂)_(n2)—CH₃. In afurther embodiment, R² is OH, R³ is H and R⁴ is H. In even a furtherembodiment, n1 is 1, 2 or 3, n2 is 9, 10, 11, 12, 13 or 14. In even afurther embodiment, n1 is 2 and n2 is 10.

In one embodiment of a compound of Formula (II), or a pharmaceuticallyacceptable salt thereof, R¹ is —C(O)—(CH₂)_(n2)—CH₃. In a furtherembodiment, R² is OH and R³ and R⁴ are H. In a further embodiment, n2 is9, 10, 11, 12, 13 or 14. In even a further embodiment, n2 is 10.

In one embodiment of a compound of Formula (II), or a pharmaceuticallyacceptable salt thereof, R¹ is —(CH₂)_(n1)—O—C(O)—(CH₂)_(n2)—CH₃ or—(CH₂)_(n1)—NH—C(O)—(CH₂)_(n2)—CH₃. In a further embodiment, R² is OH,R³ is

and R⁴ is H. In even a further embodiment, n1 is 1, 2 or 3, n2 is 10,11, 12, 13 or 14. In even a further embodiment, n1 is 2 and n2 is 10. Inyet even a further embodiment, R¹ is —(CH₂)_(n1)—O—C(O)—(CH₂)_(n2)—CH₃.

In one embodiment of a compound of Formula (II), or a pharmaceuticallyacceptable salt thereof, R¹ is —(CH₂)_(n1)—NH—C(O)—(CH₂)_(n2)—CH₃. In afurther embodiment, R² is OH, R³ is

and R⁴ is H. In even a further embodiment, n1 is 1, 2 or 3, n2 is 9, 10,11, 12, 13 or 14. In even a further embodiment, n1 is 2 and n2 is 10.

In one embodiment of a compound of Formula (II), or a pharmaceuticallyacceptable salt thereof. R¹ is —(CH₂)_(n1)—O—C(O)—(CH₂)_(n2)—CH₃. In afurther embodiment, R² is OH, R³ is

and R⁴ is H. In even a further embodiment, n1 is 1, 2 or 3, n2 is 9, 10,11, 12, 13 or 14. In even a further embodiment, n1 is 2 and n2 is 10.

In one embodiment of a compound of Formula (II), or a pharmaceuticallyacceptable salt thereof, R¹ is —(CH₂)_(n1)—C(O)—O—(CH₂)_(n2)—CH₃. In afurther embodiment, R² is OH, R³ is

and R⁴ is H. In even a further embodiment, n1 is 1, 2 or 3, n2 is 9, 10,11, 12, 13 or 14. In even a further embodiment, n1 is 2 and n2 is 10.

In one embodiment of a compound of Formula (II), or a pharmaceuticallyacceptable salt thereof, R¹ is —(CH₂)_(n1)—C(O)—NH—(CH₂)_(n2)—CH₃. In afurther embodiment, R² is OH, R³ is

and R⁴ is H. In even a further embodiment, n1 is 2 or 3, n2 is 9, 10,11, 12, 13 or 14. In even a further embodiment, n1 is 2 and n2 is 10.

In one embodiment of a compound of Formula (II), or a pharmaceuticallyacceptable salt thereof, R¹ is —C(O)—(CH₂)_(n2)—CH₃. In a furtherembodiment, R² is OH, R³ is

and R⁴ is H. In even a further embodiment, n2 is 9, 10, 11, 12, 13 or14. In even a further embodiment, n2 is 10.

In one embodiment of a compound of Formula (II), or a pharmaceuticallyacceptable salt thereof, R¹ is —(CH₂)_(n1)—O—C(O)—(CH₂)_(n2)—CH₃ or—(CH₂)_(n1)—NH—C(O)—(CH₂)_(n2)—CH₃. In a further embodiment, R² is OH,R³ is H and R⁴ is CH₂—NH—CH₂—PO₃H₂. In even a further embodiment, n1 is1, 2 or 3, n2 is 9, 10, 11, 12, 13 or 14. In even a further embodiment,n1 is 2 and n2 is 10. In yet even a further embodiment, R¹ is—(CH₂)_(n1)—O—C(O)—(CH₂)_(n2)—CH₃.

In one embodiment of a compound of Formula (II), or a pharmaceuticallyacceptable salt thereof, R¹ is —(CH₂)_(n1)—NH—C(O)—(CH₂)_(n2)—CH₃. In afurther embodiment. R² is OH, R³ is H and R⁴ is CH₂—NH—CH₂—PO₃H₂. Ineven a further embodiment, n1 is 1, 2 or 3, n2 is 9, 10, 11, 12, 13 or14. In even a further embodiment, n1 is 2 and n2 is 10.

In one embodiment of a compound of Formula (II), or a pharmaceuticallyacceptable salt thereof, R¹ is —(CH₂)_(n1)—O—C(O)—(CH₂)_(n2)—CH₃. In afurther embodiment, R² is OH, R³ is H and R⁴ is CH₂—NH—CH₂—PO₃H₂. Ineven a further embodiment, n1 is 1, 2 or 3, n2 is 9, 10, 11, 12, 13 or14. In even a further embodiment, n1 is 2 and n2 is 10.

In one embodiment of a compound of Formula (II), or a pharmaceuticallyacceptable salt thereof, R¹ is —(CH₂)_(n1)—C(O)—O—(CH₂)_(n2)—CH₃. In afurther embodiment, R² is OH, R³ is H and R⁴ is CH₂—NH—CH₂—PO₃H₂. Ineven a further embodiment, n1 is 1, 2 or 3, n2 is 9, 10, 11, 12, 13 or14. In even a further embodiment, n1 is 2 and n2 is 10.

In one embodiment of a compound of Formula (II), or a pharmaceuticallyacceptable salt thereof, R¹ is —(CH₂)_(n1)—C(O)—NH—(CH₂)_(n2)—CH₃. In afurther embodiment, R² is OH, R³ is H and R⁴ is CH₂—NH—CH₂—PO₃H₂. Ineven a further embodiment, n1 is 1, 2 or 3, n2 is 9, 10, 11, 12, 13 or14. In even a further embodiment, n1 is 2 and n2 is 10.

In one embodiment of a compound of Formula (II), or a pharmaceuticallyacceptable salt thereof, R¹ is —C(O)—(CH₂)_(n2)—CH₃. In a furtherembodiment, R² is OH, R³ is H and R⁴ is CH₂—NH—CH₂—PO₃H₂. In even afurther embodiment, n2 is 9, 10, 11, 12, 13 or 14. In even a furtherembodiment, n2 is 10.

In one embodiment of a compound of Formula (II), or a pharmaceuticallyacceptable salt thereof, R¹ is —(CH₂)_(n1)—O—C(O)—(CH₂)_(n2)—CH₃ or—(CH₂)_(n1)—NH—C(O)—(CH₂)_(n2)—CH₃. In a further embodiment, R² is—NH—(CH₂)_(q)—R⁵, R³ is H and R⁴ is H. In even a further embodiment, n1is 1, 2 or 3, n2 is 9, 10, 11, 12, 13 or 14. In even a furtherembodiment, n1 is 2 and n2 is 10. In yet even a further embodiment, R¹is —(CH₂)_(n1)—O—C(O)—(CH₂)_(n2)—CH₃. In yet even a further embodiment,q is 2 or 3 and R⁵ is N(CH₃)₂.

In one embodiment of a compound of Formula (II), or a pharmaceuticallyacceptable salt thereof, R¹ is —(CH₂)_(n1)—NH—C(O)—(CH₂)_(n2)—CH₃. In afurther embodiment, R² is —NH—(CH₂)_(q)—R⁵, R³ and R⁴ are H. In even afurther embodiment, n1 is 1, 2 or 3, n2 is 9, 10, 11, 12, 13 or 14. Ineven a further embodiment, n1 is 2 and n2 is 10. In yet even a furtherembodiment, q is 2 or 3 and R⁵ is N(CH₃)₂.

In one embodiment of a compound of Formula (II), or a pharmaceuticallyacceptable salt thereof, R¹ is —(CH₂)_(n1)—O—C(O)—(CH₂)_(n2)—CH₃. In afurther embodiment, R² is —NH—(CH₂)_(q)—R⁵, R³ and R⁴ are H. In even afurther embodiment, n1 is 1, 2 or 3, n2 is 9, 10, 11, 12, 13 or 14. Ineven a further embodiment, n1 is 2 and n2 is 10. In yet even a furtherembodiment, q is 2 or 3 and R⁵ is N(CH₃)₂.

In one embodiment of a compound of Formula (II), or a pharmaceuticallyacceptable salt thereof, R¹ is —(CH₂)_(n1)—C(O)—O—(CH₂)_(n2)—CH₃. In afurther embodiment, R² is —NH—(CH₂)_(q)—R⁵, R³ and R⁴ are H. In even afurther embodiment, n1 is 1, 2 or 3, n2 is 9, 10, 11, 12, 13 or 14. Ineven a further embodiment, n1 is 2 and n2 is 10. In yet even a furtherembodiment, q is 2 or 3 and R⁵ is N(CH₃)₂.

In one embodiment of a compound of Formula (II), or a pharmaceuticallyacceptable salt thereof, R¹ is —(CH₂)_(n1)—C(O)—NH—(CH₂)_(n2)—CH₃. In afurther embodiment, R² is —NH—(CH₂)_(q)—R⁵, R³ is H and R⁴ is H. In evena further embodiment, n1 is 1, 2 or 3, n2 is 9, 10, 11, 12, 13 or 14. Ineven a further embodiment, n1 is 2 and n2 is 10. In yet even a furtherembodiment, q is 2 or 3 and R⁵ is N(CH₃)₂.

In one embodiment of a compound of Formula (II), or a pharmaceuticallyacceptable salt thereof, R¹ is —C(O)—(CH₂)_(n2)—CH₃. In a furtherembodiment, R² is —NH—(CH₂)_(q)—R⁵, R³ is H and R⁴ is H. In even afurther embodiment, n2 is 9, 10, 11, 12, 13 or 14. In even a furtherembodiment, n2 is 10. In yet even a further embodiment, q is 2 or 3 andR⁵ is N(CH₃)₂.

In yet another embodiment, a compound of Formula (I) or (II) isprovided, wherein one or more hydrogen atoms is replaced with adeuterium atom. For example, in one embodiment of a compound of Formula(II), R³ or R⁴ is deuterium.

The compounds of present disclosure, i.e., the compounds of Formulae (I)and (II) can be prepared according to methods and steps known to thoseof ordinary skill in the art. For example, the compounds of the presentmay be prepared according to methods described in U.S. Pat. No.6,392,012; U.S. Patent Application Publication No. 2017/0152291; U.S.Patent Application Publication No. 2016/0272682, each of which is herebyincorporated by reference in their entirety for all purposes. Methodsdescribed in International Publication No. WO 2018/08197, the disclosureof which is incorporated by reference in its entirety, can also beemployed. Synthesis schemes are also provided at the Example section,herein.

Compositions provided herein can be in the form of a solution,suspension or dry powder. Compositions can be administered by techniquesknown in the art, including, but not limited to intramuscular,intravenous, intratracheal, intranasal, intraocular, intraperitoneal,subcutaneous, and transdermal routes. In addition, as discussedthroughout, the compositions can also be administered via the pulmonaryroute, e.g., via inhalation with a nebulizer, a metered dose inhaler ora dry powder inhaler.

In one embodiment, the composition provided herein comprises a pluralityof nanoparticles of the antibiotic of any of Formula (I)-(II) inassociation with a polymer. The mean diameter of the plurality ofnanoparticles, in one embodiment, is from about 50 nm to about 900 nm,for example from about 10 nm to about 800 nm, from about 100 nm to about700 nm, from about 100 nm to about 600 nm or from about 100 nm to about500 nm.

In one embodiment, the plurality of nanoparticles comprise abiodegradable polymer and the glycopeptide antibiotic of Formulae(I)-(II). In a further embodiment, the biodegradable polymer ispoly(D,L-lactide), poly(lactic acid) (PLA), poly(D,L-glycolide) (PLG),poly(lactide-co-glycolide) (PLGA), poly-(cyanoacrylate) (PCA), or acombination thereof.

In even a further embodiment, the biodegradable polymer ispoly(lactic-co-glycolitic acid) (PLGA).

Nanoparticle compositions can be prepared according to methods known tothose of ordinary skill in the art. For example, coacervation, solventevaporation, emulsification, in situ polymerization, or a combinationthereof can be employed (see, e.g., Soppimath et al. (2001). Journal ofControlled Release 70, pp. 1-20, incorporated by reference herein in itsentirety).

The amount of polymer in the composition can be adjusted, for example,to adjust the release profile of the compound of Formula (I) or (II)from the composition.

In one embodiment, a dry powder composition disclosed in one of U.S.Pat. Nos. 5,874,064, 5,855,913 and/or U.S. Patent ApplicationPublication No. 2008/0160092 is used to formulate one of theglycopeptides of Formula (I) or (II), or a pharmaceutically acceptablesalt thereof. The disclosures of U.S. Pat. Nos. 5,874,064; 5,855,913 andU.S. Patent Application Publication No. 2008/0160092 are eachincorporated by reference herein in their entireties.

In one embodiment, the composition delivered via the methods providedherein are spray dried, hollow and porous particulate compositions. Forexample, the hollow and porous particulate compositions as disclosed inWO 1999/16419, hereby incorporated in its entirety by reference for allpurposes, can be employed. Such particulate compositions compriseparticles having a relatively thin porous wall defining a large internalvoid, although, other void containing or perforated structures arecontemplated as well.

Compositions delivered via the methods provided herein, in oneembodiment, yield powders with bulk densities less than 0.5 g/cm³ or 0.3g/cm³, for example, less 0.1 g/cm3, or less than 0.05 g/cm³. Byproviding particles with very low bulk density, the minimum powder massthat can be filled into a unit dose container is reduced, whicheliminates the need for carrier particles. Moreover, the elimination ofcarrier particles, without wishing to be bound by theory, can minimizethroat deposition and any “gag” effect, since the large lactoseparticles can impact the throat and upper airways due to their size.

In some embodiments, the particulate compositions delivered via themethods provided herein comprise a structural matrix that exhibits,defines or comprises voids, pores, defects, hollows, spaces,interstitial spaces, apertures, perforations or holes. The particulatecompositions in one embodiment, are provided in a “dry” state. That is,the particulate composition possesses a moisture content that allows thepowder to remain chemically and physically stable during storage atambient temperature and easily dispersible. As such, the moisturecontent of the microparticles is typically less than 6% by weight, andfor example, less 3% by weight. In some embodiments, the moisturecontent is as low as 1% by weight. The moisture content is, at least inpart, dictated by the formulation and is controlled by the processconditions employed, e.g., inlet temperature, feed concentration, pumprate, and blowing agent type, concentration and post drying.

Reduction in bound water can lead to improvements in the dispersibilityand flowability of phospholipid based powders, leading to the potentialfor highly efficient delivery of powdered lung surfactants orparticulate composition comprising active agent dispersed in thephospholipid.

The composition administered via the methods provided herein, in oneembodiment, is a particulate composition comprising a compound ofFormula (I) or (II), or a pharmaceutically acceptable salt thereof, aphospholipid and a polyvalent cation. In particular, the compositions ofthe present invention can employ polyvalent cations inphospholipid-containing, dispersible particulate compositions forpulmonary administration to the respiratory tract for local or systemictherapy via aerosolization.

Without wishing to be bound by theory, it is thought that the use of apolyvalent cation in the form of a hygroscopic salt such as calciumchloride stabilizes a dry powder prone to moisture inducedstabilization. Without wishing to be bound by theory, it is thought thatsuch cations intercalate the phospholipid membrane, thereby interactingdirectly with the negatively charged portion of the zwitterionicheadgroup. The result of this interaction is increased dehydration ofthe headgroup area and condensation of the acyl-chain packing, all ofwhich leads to increased thermodynamic stability of the phospholipids.Other benefits of such dry powder compositions are provided in U.S. Pat.No. 7,442,388, the disclosure of which is incorporated herein in itsentirety for all purposes.

The polyvalent cation for use in the present invention in oneembodiment, is a divalent cation. In a further embodiment, the divalentcation is calcium, magnesium, zinc or iron. The polyvalent cation ispresent in one embodiment, to increase the Tm of the phospholipid suchthat the particulate composition exhibits a Tm which is greater than itsstorage temperature Ts by at least 20° C. The molar ratio of polyvalentcation to phospholipid in one embodiment, is 0.05, e.g., from about 0.05to about 2.0, or from about 0.25 to about 1.0. In one embodiment, themolar ratio of polyvalent cation to phospholipid is about 0.50. In oneembodiment, the polyvalent cation is calcium and is provided as calciumchloride.

According to one embodiment, the phospholipid is a saturatedphospholipid. In a further embodiment, the saturated phospholipid is asaturated phosphatidylcholine. Acyl chain lengths that can be employedrange from about C₁₆ to C₂₂. For example, in one embodiment an acylchain length of 16:0 or 18:0 (i.e., palmitoyl and stearoyl) is employed.In one phospholipid embodiment, a natural or synthetic lung surfactantis provided as the phospholipid component. In this embodiment, thephospholipid can make up to 90 to 99.9% w/w of the lung surfactant.Suitable phospholipids according to this aspect of the invention includenatural or synthetic lung surfactants such as those commerciallyavailable under the trademarks ExoSurf, InfaSurf® (Ony, Inc.), Survanta,CuroSurf, and ALEC.

The Tm of the phospholipid-glycopeptide particles, in one embodiment, ismanipulated by varying the amount of polyvalent cations in thecomposition.

Phospholipids from both natural and synthetic sources are compatiblewith the compositions administered by the methods provided herein, andmay be used in varying concentrations to form the structural matrix.Generally compatible phospholipids comprise those that have a gel toliquid crystal phase transition greater than about 40° C. Theincorporated phospholipids in one embodiment, are relatively long chain(i.e., C₁₆-C₂₂) saturated lipids and in a further embodiment, comprisesaturated phospholipids. In even a further embodiment, the saturatedphospholipid is a saturated phosphatidylcholine. In even a furtherembodiment, the saturated phosphatidylcholine has an acyl chain lengthsof 16:0 or 18:0 (palmitoyl or stearoyl). Exemplary phospholipids usefulin the disclosed stabilized preparations comprise, phosphoglyceridessuch as dipalmitoylphosphatidylcholine (DPPC),disteroylphosphatidylcholine (DSPC), diarachidoylphosphatidylcholinedibehenoylphosphatidylcholine, diphosphatidyl glycerol, short-chainphosphatidylcholines, long-chain saturated phosphatidylethanolamines,long-chain saturated phosphatidylserines, long-chain saturatedphosphatidylglycerols, long-chain saturated phosphatidylinositols.

In addition to the phospholipid, a co-surfactant or combinations ofsurfactants, including the use of one or more in the liquid phase andone or more associated with the particulate compositions can be used inthe compositions delivered via the methods provided herein. By“associated with or comprise” it is meant that the particulatecompositions may incorporate, adsorb, absorb, be coated with or beformed by the surfactant. Surfactants include fluorinated andnonfluorinated compounds and can include saturated and unsaturatedlipids, nonionic detergents, nonionic block copolymers, ionicsurfactants and combinations thereof. In one embodiment comprisingstabilized dispersions, nonfluorinated surfactants are relativelyinsoluble in the suspension medium.

Compatible nonionic detergents suitable as co-surfactants in thecompositions provided herein include sorbitan esters including sorbitantrioleate (Span™ 85), sorbitan sesquioleate, sorbitan monooleate,sorbitan monolaurate, polyoxyethylene (20) (Brij® S20), sorbitanmonolaurate, and polyoxyethylene (20) sorbitan monooleate, oleylpolyoxyethylene (2) ether, stearyl polyoxyethylene (2) ether, laurylpolyoxyethylene (4) ether, glycerol esters, and sucrose esters. Blockcopolymers include diblock and triblock copolymers of polyoxyethyleneand polyoxypropylene, including poloxamer 188 (Pluronic® F-68),poloxamer 407 (Pluronic® F-127), and poloxamer 338. Ionic surfactantssuch as sodium sulfosuccinate, and fatty acid soaps may also beutilized.

The phospholipid-glycopeptide particulate compositions can includeadditional lipids such as a glycolipid, ganglioside GM1, sphingomyelin,phosphatidic acid, cardiolipin; a lipid bearing a polymer chain such aspolyethylene glycol, chitin, hyaluronic acid, or polyvinylpyrrolidone; alipid bearing sulfonated mono-, di-, and polysaccharides; a fatty acidsuch as palmitic acid, stearic acid, and/or oleic acid; cholesterol,cholesterol esters, and cholesterol hemisuccinate.

In addition to the phospholipid and polyvalent cation, the particulatecomposition delivered via the methods provided herein can also include abiocompatible, and in some embodiments, biodegradable polymer,copolymer, or blend or other combination thereof. The polymer in oneembodiment is a polylactide, polylactide-glycolide, cyclodextrin,polyacrylate, methylcellulose, carboxymethylcellulose, polyvinylalcohol, polyanhydride, polylactam, polyvinyl pyrrolidone,polysaccharide (e.g., dextran, starch, chitin, chitosan), hyaluronicacid, protein (e.g., albumin, collagen, gelatin, etc.).

Besides the aforementioned polymer materials and surfactants, otherexcipients can be added to a particulate composition, for example, toimprove particle rigidity, production yield, emitted dose anddeposition, shelf-life and/or patient acceptance. Such optionalexcipients include, but are not limited to: coloring agents, tastemasking agents, buffers, hygroscopic agents, antioxidants, and chemicalstabilizers. Other excipients may include, but are not limited to,carbohydrates including monosaccharides, disaccharides andpolysaccharides. For example, monosaccharides such as dextrose(anhydrous and monohydrate), galactose, mannitol, D-mannose, sorbitol,sorbose and the like; disaccharides such as lactose, maltose, sucrose,trehalose, and the like; trisaccharides such as raffinose and the like;and other carbohydrates such as starches (hydroxyethylstarch),cyclodextrins and maltodextrins. Mixtures of carbohydrates and aminoacids are further held to be within the scope of the present invention.The inclusion of both inorganic (e.g., sodium chloride), organic acidsand their salts (e.g., carboxylic acids and their salts such as sodiumcitrate, sodium ascorbate, magnesium gluconate, sodium gluconate,tromethamine hydrochloride, etc.) and buffers can also be undertaken.Salts and/or organic solids such as ammonium carbonate, ammoniumacetate, ammonium chloride or camphor can also be employed.

According to one embodiment, the particulate compositions may be used inthe form of dry powders or in the form of stabilized dispersionscomprising a non-aqueous phase. The dispersions or powders of thepresent invention may be used in conjunction with metered dose inhalers(MDIs), dry powder inhalers (DPIs), atomizers, or nebulizers to providefor pulmonary delivery.

While several procedures are generally compatible with making certaindry powder compositions described herein, spray drying is a particularlyuseful method. As is well known, spray drying is a one-step process thatconverts a liquid feed to a dried particulate form. With respect topharmaceutical applications, it will be appreciated that spray dryinghas been used to provide powdered material for various administrativeroutes including inhalation. See, for example, M. Sacchetti and M. M.Van Oort in: Inhalation Aerosols: Physical and Biological Basis forTherapy, A. J. Hickey, ed. Marcel Dekkar, New York, 1996, which isincorporated herein by reference in its entirety for all purposes. Ingeneral, spray drying consists of bringing together a highly dispersedliquid, and a sufficient volume of hot air to produce evaporation anddrying of the liquid droplets. The preparation to be spray dried or feed(or feed stock) can be any solution, suspension, slurry, colloidaldispersion, or paste that may be atomized using the selected spraydrying apparatus. In one embodiment, the feed stock comprises acolloidal system such as an emulsion, reverse emulsion, microemulsion,multiple emulsion, particulate dispersion, or slurry. Typically, thefeed is sprayed into a current of warm filtered air that evaporates thesolvent and conveys the dried product to a collector. The spent air isthen exhausted with the solvent.

It will further be appreciated that spray dryers, and specifically theiratomizers, may be modified or customized for specialized applications,e.g., the simultaneous spraying of two solutions using a double nozzletechnique. More specifically, a water-in-oil emulsion can be atomizedfrom one nozzle and a solution containing an anti-adherent such asmannitol can be co-atomized from a second nozzle. In one embodiment, itmay be desirable to push the feed solution though a custom designednozzle using a high pressure liquid chromatography (HPLC) pump. Examplesof spray drying methods and systems suitable for making the dry powdersof the present invention are disclosed in U.S. Pat. Nos. 6,077,543,6,051,256, 6,001,336, 5,985,248, and 5,976,574, each of which isincorporated in their entirety by reference.

While the resulting spray-dried powdered particles typically areapproximately spherical in shape, nearly uniform in size and frequentlyare hollow, there may be some degree of irregularity in shape dependingupon the incorporated glycopeptide of Formulae (I)-(II) and the spraydrying conditions. In one embodiment, an inflating agent (or blowingagent) is used in the spray-dried powder production, e.g., as disclosedin WO 99/16419, incorporated by reference herein in its entirety for allpurposes. Additionally, an emulsion can be included with the inflatingagent as the disperse or continuous phase. The inflating agent can bedispersed with a surfactant solution, using, for instance, acommercially available microfluidizer at a pressure of about 5000 to15,000 PSI. This process forms an emulsion, and in some embodiments, anemulsion stabilized by an incorporated surfactant, and can comprisesubmicron droplets of water immiscible blowing agent dispersed in anaqueous continuous phase. The blowing agent in one embodiment, is afluorinated compound (e.g., perfluorohexane, perfluorooctyl bromide,perfluorooctyl ethane, perfluorodecalin, perfluorobutyl ethane) whichvaporizes during the spray-drying process, leaving behind generallyhollow, porous aerodynamically light microspheres. Other suitable liquidblowing agents include nonfluorinated oils, chloroform, Freons, ethylacetate, alcohols and hydrocarbons. Nitrogen and carbon dioxide gasesare also contemplated as a suitable blowing agent. Perfluorooctyl ethaneis the blowing agent, in one embodiment.

Whatever components are selected, the first step in particulateproduction in one embodiment, comprises feed stock preparation. Theselected glycopeptide is dissolved in a solvent, for example water,dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetonitrile,ethanol, methanol, or combinations thereof, to produce a concentratedsolution. The polyvalent cation may be added to the glycopeptidesolution or may be added to the phospholipid emulsion as discussedbelow. The glycopeptide may also be dispersed directly in the emulsion,particularly in the case of water insoluble agents. Alternatively, theglycopeptide is incorporated in the form of a solid particulatedispersion. The concentration of the glycopeptide used is dependent onthe amount of glycopeptide required in the final powder and theperformance of the delivery device employed (e.g., the fine particledose for a MDI or DPI). As needed, cosurfactants such as poloxamer 188or span 80 may be dispersed into this annex solution. Additionally,excipients such as sugars and starches can also be added.

In one embodiment, a polyvalent cation-containing oil-in-water emulsionis then formed in a separate vessel. The oil employed in one embodiment,is a fluorocarbon (e.g., perfluorooctyl bromide, perfluorooctyl ethane,perfluorodecalin) which is emulsified with a phospholipid. For example,polyvalent cation and phospholipid may be homogenized in hot distilledwater (e.g., 60° C.) using a suitable high shear mechanical mixer (e.g.,Ultra-Turrax model T-25 mixer) at 8000 rpm for 2 to 5 minutes. In oneembodiment, 5 to 25 g of fluorocarbon is added dropwise to the dispersedsurfactant solution while mixing. The resulting polyvalentcation-containing perfluorocarbon in water emulsion is then processedusing a high pressure homogenizer to reduce the particle size. In oneembodiment, the emulsion is processed at 12,000 to 18,000 PSI, 5discrete passes and kept at 50 to 80° C.

The glycopeptide solution (or suspension) and perfluorocarbon emulsionare then combined and fed into the spray dryer. In one embodiment, thetwo preparations are miscible. While the glycopeptide is solubilizedseparately for the purposes of the instant discussion it will beappreciated that, in other embodiments, the glycopeptide may besolubilized (or dispersed) directly in the emulsion. In such cases, theglycopeptide emulsion is simply spray dried without combining a separateglycopeptide preparation.

Operating conditions such as inlet and outlet temperature, feed rate,atomization pressure, flow rate of the drying air, and nozzleconfiguration can be adjusted in accordance with the manufacturer'sguidelines in order to produce the desired particle size, and productionyield of the resulting dry particles. The selection of appropriateapparatus and processing conditions are well within the purview of askilled artisan. In one embodiment, the particulate compositioncomprises hollow, porous spray dried micro- or nano-particles.

Along with spray drying, particulate compositions useful in the presentinvention may be formed by lyophilization. Those skilled in the art willappreciate that lyophilization is a freeze-drying process in which wateris sublimed from the composition after it is frozen. Methods forproviding lyophilized particulates are known to those of skill in theart. The lyophilized cake containing a fine foam-like structure can bemicronized using techniques known in the art.

Besides the aforementioned techniques, the glycopeptide particulatecompositions or glycopeptide particles provided herein may also beformed using a method where a feed solution (either emulsion or aqueous)containing wall forming agents is rapidly added to a reservoir of heatedoil (e.g., perflubron or other high boiling FCs) under reduced pressure.The water and volatile solvents of the feed solution rapidly boils andare evaporated. This process provides a perforated structure from thewall forming agents similar to puffed rice or popcorn. In oneembodiment, the wall forming agents are insoluble in the heated oil. Theresulting particles can then be separated from the heated oil using afiltering technique and subsequently dried under vacuum.

In another embodiment, the particulate compositions of the presentinvention may also be formed using a double emulsion method. In thedouble emulsion method, the medicament is first dispersed in a polymerdissolved in an organic solvent (e.g., methylene chloride, ethylacetate) by sonication or homogenization. This primary emulsion is thenstabilized by forming a multiple emulsion in a continuous aqueous phasecontaining an emulsifier such as polyvinylalcohol. Evaporation orextraction using conventional techniques and apparatus then removes theorganic solvent. The resulting particles are washed, filtered and driedprior to combining them with an appropriate suspension medium.

In order to maximize dispersibility, dispersion stability and optimizedistribution upon administration, the mean geometric particle size ofthe particulate compositions in one embodiment, is from about 0.5-50 μm,for example from about 0.5 μm to about 10 μm or from about 0.5 to about5 μm. In one embodiment, the mean geometric particle size (or diameter)of the particulate compositions is less than 20 μm or less than 10 μm.In a further embodiment, the mean geometric diameter is ≤about 7 μm or≤5 μm. In even a further embodiment, the mass geometric diameter is≤about 2.5 μm. In one embodiment, the particulate composition comprisesa powder of dry, hollow, porous spherical shells of from about 0.1 toabout 10 μm, e.g., from about 0.5 to about 5 μm in diameter, with shellthicknesses of approximately 0.1 μm to approximately 0.5 μm.

Methods for treating infectious diseases, especially those caused byGram-positive microorganisms, are provided. The method comprises, in oneembodiment, administering to a patient in need of treatment, acomposition comprising an effective amount of an LGPC derivative, or apharmaceutically acceptable salt thereof. The LGPC derivative, containsa primary amino conjugated lipophilic moiety that is cleavable byenzymatic hydrolysis. The lipophilic moiety is conjugated to the primaryamino group via a functional group that is capable of undergoingenzymatic hydrolysis. The functional group that undergoes enzymatichydrolysis, in one embodiment, in conjugated to the primary amino groupvia a straight chain or branched alkyl group, e.g., a methyl, ethyl,propyl or butyl group. In another embodiment, the functional group is anamide that comprises the nitrogen atom from the primary amino group ofthe glycopeptide. The method comprises, in one embodiment, administeringthe composition comprising the LGPC derivative to the patient in need oftreatment via inhalation.

In one embodiment of the methods provided herein, a compositioncomprising an effective amount of a compound of Formula (I) or (II), ora pharmaceutically acceptable salt of one of the foregoing, isadministered to a patient in need of treatment.

Without wishing to be bound by a particular theory, it is believed thatthe R¹ groups conjugated to the glycopeptides provided herein facilitatecellular uptake of the glycopeptide at the site of infection, forexample, macrophage uptake.

An “effective amount” of a compound of Formula (I) or (II) or apharmaceutically acceptable salt of Formula (I) or (II), is an amountthat can provide the desired therapeutic response. The effective amountcan refer to a single dose as part of multiple doses during anadministration period, or as the total dosage of the LGPC given duringan administration period. A treatment regimen can include substantiallythe same dose for each LGPC administration, or can comprise at leastone, at least two or at least three different dosages.

According to one embodiment, a method is provided to treat an infectiondue to a Gram-positive bacterium, including, but not limited to, generaStaphylococcus, Streptococcus, Enterococcus, Bacillus, Corynebacleriutn,Nocardia, Clostridium, and Listeria. In one embodiment, the infection isdue to a Gram-positive cocci bacterium. In a further embodiment, theGram-positive cocci infection is a Staphylococcus, Enterococcus orStreptococcus infection.

The bacterial infection treated by the methods provided herein may bepresent as planktonic free-floating bacteria, a biofilm, or acombination thereof. In one embodiment, the infection treated with themethods provided herein is a pulmonary infection.

In one embodiment, the bacterial infection is a Gram-positive bacterialinfection. In a further embodiment, the bacterial infection is apulmonary Gram-positive bacterial infection.

In one embodiment, the Gram-positive bacterial infection is aGram-positive cocci infection. In a further embodiment, theGram-positive cocci infection is a Streptococcus, Enterococcus or aStaphylococcus infection.

Over the past few decades, there has been a decrease in thesusceptibility of Gram-positive cocci to antibacterials for thetreatment of infection. See, e.g., Alvarez-Lerma et al. (2006) Drugs 66,pp. 751-768, incorporated by reference herein in its entirety for allpurposes. As such, in one aspect, the present invention addresses thisneed by providing a composition comprising an effective amount of acompound of Formula (I) or (II), or a pharmaceutically acceptable saltthereof, in a method for treating a patient in need thereof for aGram-positive cocci infection that is resistant to a differentantibacterial. For example, in one embodiment, the Gram-positive cocciinfection is a penicillin resistant or a vancomycin resistant bacterialinfection. In a further embodiment, the resistant bacterial infection isa methicillin-resistant Staphylococcus infection, e.g.,methicillin-resistant S. aureus or a methicillin-resistantStaphylococcus epidermidis infection. In another embodiment, theresistant bacterial infection is an oxacillin-resistant Staphylococcus(e.g., S. aureus) infection, a vancomycin-resistant Enterococcusinfection or a penicillin-resistant Streptococcus (e.g., S. pneumoniae)infection. In yet another embodiment, the Gram-positive cocci infectionis a vancomycin-resistant enterococci (VRE), methicillin-resistantStaphylococcus aureus (MRSA), methicillin-resistant Staphylococcusepidermidis (MRSE), vancomycin resistant Enterococcus faecium alsoresistant to teicoplanin (VRE Fm Van A), vancomycin resistantEnterococcus faecium sensitive to teicoplanin (VRE Fm Van B), vancomycinresistant Enterococcus faecalis also resistant to teicoplanin (VRE FsVan A), vancomycin resistant Enterococcus faecalis sensitive toteicoplanin (VRE Fs Van B), or penicillin-resistant Streptococcuspneumoniae (PSRP).

According to one embodiment, a method is provided for treating abacterial infection comprising administering a composition comprising aneffective amount of a compound of Formula (I) or (II), or apharmaceutically-acceptable salt thereof, to the patient. For example,the composition can be administered to the patient via pulmonaryadministration or via parenteral administration (e.g., intravenous).

As provided herein, LGPC derivatives of Formulae (I) and (II) areprovided. Such compounds are useful in the treatment of bacterialinfections, including, but not limited to, pulmonary infections, andspecifically, pulmonary infections caused by Gram-positive bacteria. TheLGPC derivatives provided herein possess a biologically-labile moiety(e.g., amide, ester) connected via an amine group of the glycopeptide,e.g., a primary amine. Subsequent to administration, thebiologically-labile moiety undergoes cleavage by any available mechanism(e.g., hydrolysis or enzymatic cleavage), providing one or moreglycopeptide metabolites. In some embodiments, the glycopeptidemetabolite provides a decreased residence time in the lungs compared tothe unmetabolized glycopeptide compound, thereby assisting inelimination of the therapeutic agent from this organ.

In one embodiment, the compound of Formula (I) or (II), and itsrespective metabolite, provide a synergistic effect against thebacterial infection being treated.

Metabolites of LGPC derivatives of Formula (I) (or a pharmaceuticallyacceptable salt thereof), in one embodiment, have the followingstructures (Glycopeptide, R¹, n1 and n2 as defined above).

-   -   Glycopeptide-(CH₂)_(n1)—OH (a metabolite of a compound of        Formula (I), where R¹ is —(CH₂)_(n1)—O—C(O)—(CH₂)_(n2)—CH₃;        —(CH₂)_(n1)—O—C(O)—O—(CH₂)_(n2)—CH₃; or        —(CH₂)_(n1)—O—C(O)—NH—(CH₂)_(n2)—CH₃)    -   Glycopeptide-(CH₂)_(n1)—NH₂ (metabolite of a compound of Formula        (I), where R¹ is —(CH₂)_(n1)—NH—C(O)—(CH₂)_(n2)—CH₃; or        —(CH₂)_(n1)—NH—C(O)—O—(CH₂)_(n2)—CH₃)    -   Glycopeptide-(CH₂)_(n1)—C(O)OH (a metabolite of a compound of        Formula (I), where R¹ is —(CH₂)_(n1)—C(O)—NH—(CH₂)_(n2)—CH₃ or        —(CH₂)_(n1)—C(O)—O—(CH₂)_(n2)—CH₃)

Metabolites of LGPC derivatives of Formula (II) (or a pharmaceuticallyacceptable salt thereof), have the following structures (R¹, R², R³, R⁴,n1 and n2 defined above):

In one embodiment, a Gram-positive cocci infection is treated with oneof the methods provided herein. In a further embodiment, theGram-positive cocci infection is a Staphylococcus infection.Staphylococcus is Gram-positive non-motile bacteria that colonizes skinand mucus membranes. Staphylococci are spherical and occur inmicroscopic clusters resembling grapes. The natural habitat ofStaphylococcus is nose; it can be isolated in 50% of normal individuals.20% of people are skin carriers and 10% of people harbor Staphylococcusin their intestines. Examples of Staphylococci infections treatable withthe methods and compositions provided herein, include S. aureus, S.epidermidis, S. auricularis, S. carnosus, S. haemolyticus, S. hyicus, S.intermedius, S. lugdunensis, S. saprophytics, S. sciuri, S. simulans,and S. wameri. In one embodiment, the Staphylococcus infection is aStaphylococcus aureus (S. aureus) infection.

While there have been about 20 species of Staphylococcus reported, onlyStaphylococcus aureus and Staphylococcus epidermis are known to besignificant in their interactions with humans.

In one embodiment, the Staphylococcus infection is a Staphylococcushaemolyticus (S. haemolyticus) infection. In another embodiment, theStaphylococcus infection is a Staphylococcus epidermis (S. epidermis)infection. A Staphylococcus infection, e.g., S. aureus is treated in oneembodiment, in a patient that has been diagnosed with mechanicalventilation-associated pneumonia.

In one embodiment, the S. aureus infection is a methicillin-resistantStaphylococcus aureus (MRSA) infection. In another embodiment, the S.aureus infection is a methicillin-sensitive S. aureus (MSSA) infection.In another embodiment, the S. aureus infection is a S. aureus (VISA)infection, or a vancomycin-resistant S. aureus (VRSA) infection.

In one embodiment, the Staphylococcus species is resistant to apenicillin such as methicillin. In a further embodiment, theStaphylococcus species is methicillin-resistant Staphylococcus aureus(MRSA) or methicillin-resistant Staphylococcus epidermidis (MRSE). TheStaphylococcus species, in another embodiment, is methicillin-sensitiveS. aureus (MSSA), vancomycin-intermediate S. aureus (VISA), orvancomycin-resistant S. aureus (VRSA).

S. aureus colonizes mainly the nasal passages, but it may be foundregularly in most anatomical locales, including skin oral cavity, andgastrointestinal tract. In one embodiment, a S. aureus infection istreated with one of the methods and/or compositions provided herein.

The S. aureus infection can be a healthcare associated, i.e., acquiredin a hospital or other healthcare setting, or community-acquired.

In one embodiment, the Staphylococcal infection treated with one of themethods and/or compositions provided herein, causes endocarditis orsepticemia (sepsis). As such, the patient in need of treatment with oneof the methods and/or compositions provided herein, in one embodiment,is an endocarditis patient. In another embodiment, the patient is asepticemia (sepsis) patient.

In one embodiment, the bacterial infection is erythromycin-resistant(erm^(R)), vancomycin-intermediate S. aureus (VISA) heterogeneousvancomycin-intermediate S. aureus (hVISA), S. epidermidiscoagulase-negative staphylococci (CoNS), penicillin-intermediate S.pneumoniae (PISP), or penicillin-resistant S. pneumoniae (PRSP). In evena further embodiment, the administering comprises administering viainhalation.

In one embodiment, the Gram-positive cocci infection is a Streptococcusinfection. Streptococci are Gram-positive, non-motile cocci that dividein one plane, producing chains of cells. The primary pathogens includeS. pyrogenes and S. pneumoniae but other species can be opportunistic.S. pyrogenes is the leading cause of bacterial pharyngitis andtonsillitis. It can also produce sinusitis, otitis, arthritis, and boneinfections. Some strains prefer skin, producing either superficial(impetigo) or deep (cellulitis) infections. Streptococcus pnemoniae istreated, in one embodiment, in a patient that has been diagnosed withcommunity-acquired pneumonia or purulent meningitis.

S. pneumoniae is the major cause of bacterial pneumonia in adults, andin one embodiment, an infection due to S. pneumoniae is treated via oneof the methods and/or compositions provided herein. Its virulence isdictated by its capsule. Toxins produced by streptococci include:streptolysins (S & O), NADase, hyaluronidase, streptokinase, DNAses,erythrogenic toxin (which causes scarlet fever rash by producing damageto blood vessels; requires that bacterial cells are lysogenized by phagethat encodes toxin). Examples of Streptococcus infections treatable withthe compositions and methods provided herein include, S. agalactiae, S.anginosus, S. bovis, S. canis, S. constellatus, S. dysgalactiae, S.equi, S. equinus, S. mae, S. intermedins, S. mitis, S. mutans, S.oralis, S. parasanguinis, S. peroris, S. pneumoniae, S. pyogenes, S.ratti, S. salivarius, S. salivarius ssp. thermophilies, S. sanguinis, S.sobrinus, S. suis, S. uteris, S. vestibularis, S. viridans, and S.zooepidemicus.

In one embodiment, the Streptococcus infection is a S. pyogenes, S.agalactiae, S. dysgalactiae, S. bovis, S. anginosus, S. sanguinis, S.suis, S. mitis, S. pneumoniae, or a S. mutans infection. In anotherembodiment, the Streptococcus infection is a S. mutans infection. Instill another embodiment, the Streptococcus infection is a S. pneumoniaeinfection. In yet another embodiment, the Streptococcus infection is aS. dysgalactiae infection. In a further embodiment, the Streptococcusinfection is a S. pyogenes infection.

In one embodiment, the Gram-positive cocci infection is an Enterococcusinfection. In another embodiment, the Enterococcus infection is avancomycin resistant infection (VRE). In a further embodiment, theEnterococcus infection is a vancomycin sensitive infection (VSE).

The genus Enterococci consists of Gram-positive, facultatively anaerobicorganisms that are ovoid in shape and appear on smear in short chains,in pairs, or as single cells. Enterococci are important human pathogensthat are increasingly resistant to antimicrobial agents. Examples ofEnterococci treatable with the methods and compositions provided hereinare E. avium, E. durans, E. faecalis, E. faecium, E. gallinarum, and E.solitarius. An Enterococcus species is treated, in one embodiment, in apatient that has been diagnosed with a urinary-catheter relatedinfection.

In one embodiment of the methods provided herein, a patient in needthereof is treated for an Enterococcus faecalis (E. faecalis) infection.In a further embodiment, the infection is a pulmonary infection. Inanother embodiment, a patient in need thereof is treated for anEnterococcus faecium (E. faecium) infection. In a further embodiment,the infection is a pulmonary infection.

In one embodiment, a patient in need thereof is treated for anEnterococcus infection that is resistant or sensitive to vancomycin orresistant or sensitive to penicillin. In a further embodiment, theEnterococcus infection is an E. faecalis or E. faecium infection. In aspecific embodiment, the Enterococcus infection is an Enterococcusfaecalis (E. faecalis) infection. In one embodiment, the E. faecalisinfection is a vancomycin-sensitive E. faecalis infection. In anotherembodiment, the E. faecalis infection is a vancomycin-resistant E.faecalis infection. In yet another embodiment, the E. faecalis infectionis an ampicillin-resistant E. faecalis infection. In another embodiment,the Enterococcus infection is an Enterococcus faecium (E. faecium)infection In still another embodiment, the E. faecium infection is avancomycin-resistant E. faecium infection. In a further embodiment, theE. faecium infection is an ampicillin-resistant E. faecium infection. Inyet a further embodiment, the E. faecium infection is avancomycin-sensitive E. faecium infection.

Bacteria of the genus Bacillus are aerobic, endospore-forming,Gram-positive rods, and infections due to such bacteria are treatablevia the methods and compositions provided herein. Bacillus species canbe found in soil, air, and water where they are involved in a range ofchemical transformations. In one embodiment, a method is provided hereinto treat a Bacillus anthracis (B. anthracis) infection with aglycopeptide composition. Bacillus anthracis, the infection that causesAnthrax, is acquired via direct contact with infected herbivores orindirectly via their products. The clinical forms include cutaneousanthrax, from handling infected material, intestinal anthrax, fromeating infected meat, and pulmonary anthrax from inhaling spore-ladendust. The route of administration of the glycopeptide will varydepending on how the patient acquires the B. anthracis infection. Forexample, in the case of pulmonary anthrax, the patient, in oneembodiment, is treated via a dry powder inhaler, nebulizer or metereddose inhaler.

Several other Bacillus species, in particular, B. cereus, B. subtilisand B. licheniformis, are associated periodically withbacteremia/septicemia, endocarditis, meningitis, and infections ofwounds, the ears, eyes, respiratory tract, urinary tract, andgastrointestinal tract, and are therefore treatable with the methods andcompositions provided herein. Examples of pathogenic Bacillus specieswhose infection is treatable with the methods and compositions providedherein, include, but are not limited to, B. anthracis, B. cereus and B.coagulans.

Corynebacteria are small, generally non-motile, Gram-positive, nonsporalating, pleomorphic bacilli and infections due to these bacteriaare treatable via the methods provided herein. Corybacterium diphtheriais the etiological agent of diphtheria, an upper respiratory diseasemainly affecting children, and is treatable via the methods andcompositions provided herein. Examples of other Corynebacteria speciestreatable with the methods and compositions provided herein includeCorynebacterium diphtheria, Corynebacterium pseudotuberculosis,Corynebacterium tenuis, Corynebacterium striatum, and Corynebacteriumminutissimum.

The bacteria of the genus Nocardia are Gram-positive, partiallyacid-fast rods, which grow slowly in branching chains resembling fungalhyphae. Three species cause nearly all human infections: N. asteroides,N. brasiliensis, and N. caviae, and patients with such infections can betreated with the compositions and methods provided herein. Infection isby inhalation of airborne bacilli from an environmental source (soil ororganic material). Other Nocardial species treatable with the methodsand compositions provided herein include N. aerocolonigenes, N.africana, N. argentinensis, N. asteroides, N. blackwellu, N.brasiliensis, N. brevicalena, N. cornea, N. caviae, N. cerradoensis, N.corallina, N. cyriacigeorgica, N. dassonvillei, N. elegans, N.farcinica, N. nigiitansis, N. nova, N. opaca, N. otitidis-cavarium, N.paucivorans, N. pseudobrasiliensis, N. rubra, N. transvelencesis, N.uniformis, N. vaccinii, and N. veterana.

Clostridia are spore-forming, Gram-positive anaerobes, and infectionsdue to such bacteria are treatable via the methods and compositionsprovided herein. In one embodiment, one of the methods provided hereinare used to treat a Clostridium tetani (C. tetani) infection, theetiological agent of tetanus. In another embodiment, one of the methodsprovided herein is used to treat a Clostridium botidinum (C. botidinum)infection, the etiological agent of botulism. In yet another embodiment,one of the methods provided herein is used to treat a C. perfringensinfection, one of the etiological agents of gas gangrene. OtherClostridium species treatable with the methods and compositions of thepresent invention, include, C. difficile, C. perfringens, and/or C.sordellii. In one embodiment, the infection to be treated is a C.difficile infection.

Listeria are non-spore-forming, nonbranching Gram-positive rods thatoccur individually or form short chains. Listeria monocytogenes (L.monocytogenes) is the causative agent of listeriosis, and in oneembodiment, a patient infected with L. monocytogenes is treated with oneof the methods and compositions provided herein. Examples of Listeriaspecies treatable with the methods and compositions provided herein,include L. grayi, L. innocua, L. ivanovii, L. monocytogenes, L.seeligeri, L. murrayi, and L. welshimeri.

In some embodiments, the methods disclosed herein are useful in treatingGram-negative infections. In one embodiment, the bacterial infection isa Burkholderia infection. In some embodiments, the Burkholderiainfection is a Burkholderia pseudomallei (B. pseudomallei), B. dolosa,B. fungorum, B. gladioli, B. multivorans, B. vietnamiensis, B.ambifaria, B. andropogonis, B. anthina, B. brasilensis, B. calcdonica,B. caribensis or a B. caryophylli infection.

Burkholderia is a genus of Proteobacteria whose pathogenic membersinclude among other the Burkholderia cepacia complex which attackshumans; Burkholderia pseudomallei, causative agent of melioidosis; andBurkholderia cepacia, an important pathogen of pulmonary infections inpeople with cystic fibrosis. The Burkholderia (previously part ofPseudomonas) genus name refers to a group of virtually ubiquitousGram-negative, obligately aerobic, rod-shaped bacteria that are motileby means of single or multiple polar flagella, with the exception ofBurkholderia mallei which is nonmotile.

In other embodiment, the bacterial infection is a Yersinia pestis (Y.pestis) infection.

Yersinia pestis (formerly Pasteurella pestis) is a Gram-negative,rod-shaped coccobacillus, non-mobile with no spores. It is a facultativeanaerobic organism that can infect humans via the oriental rat flea. Itcauses the disease plague, which takes three main forms: pneumonic,septicemic, and bubonic plagues.

In yet another embodiment, the bacterial infection is a Francisellatularensis (F. tularensis) infection. Francisella tularensis is apathogenic species of Gram-negative, rod-shaped coccobacillus, an aerobebacterium. It is non-spore forming, non-motile and the causative agentof tularemia, the pneumonic form of which is often lethal withouttreatment. It is a fastidious, facultative intracellular bacterium whichrequires cysteine for growth.

The bacterial infection in one embodiment, is a respiratory tractinfection. In a further embodiment, the infection is a resistantbacterial infection, for example, one of the infections provided above.The patient treatable by the methods and compositions provided herein,in one embodiment, has been diagnosed with a community-acquiredrespiratory tract infection, for example, pneumonia. In one embodiment,the bacterial infection treated in the pneumonia patient is a S.pneumoniae infection. In another embodiment, the bacterial infectiontreated in the pneumonia patient is Mycoplasma pneumonia or a Legionellaspecies. In another embodiment, the bacterial infection in the pneumoniapatient is penicillin resistant, e.g., penicillin-resistant S.pneumoniae.

The bacterial infection, in one embodiment, is a hospital acquiredinfection (HAI), or acquired in another health care facility, e.g., anursing home, rehabilitation facility, outpatient clinic, etc. Suchinfections are also referred to as nosocomial infections. In a furtherembodiment, the bacterial infection is a respiratory tract infection ora skin infection. In one embodiment, the HAI is pneumonia. In a furtherembodiment, the pneumonia is due to S. aureus, e.g., MRSA.

Respiratory infections and in particular pulmonary infections are quiteproblematic for patients afflicted with cystic fibrosis (CF). In fact,such infections are the main cause of pulmonary deterioration in thispopulation of patients. The lungs of CF patients are colonized andinfected by bacteria from an early age. These bacteria thrive in thealtered mucus, which collects in the small airways of the lungs. Theformation of biofilms makes infections of this origin difficult totreat. Consequently, more robust treatments options are needed. Thus, inone embodiment, the methods disclosed herein are useful in treating apatient with cystic fibrosis having a bacterial infection. In someembodiments, the bacterial infection is a pulmonary infection. In otherembodiments, the pulmonary infection is comprised of a biofilm.

With respect to pulmonary infections, the compounds and compositionsprovided herein can be delivered to a patient in need of treated via aninhalation delivery device that provides local administration to thesite of infection.

The inhalation delivery device employed in embodiments of the methodsprovided herein can be a nebulizer, dry powder inhaler (DPI), or ametered dose inhaler (MDI), or any other suitable inhalation deliverydevice known to one of ordinary skill in the art. The device can containand be used to deliver a single dose of the composition or the devicecan contain and be used to deliver multi-doses of the composition of thepresent invention.

According to one embodiment, a dry powder particulate composition isdelivered to a patient in need thereof via a metered dose inhaler (MDI),dry powder inhaler (DPI), atomizer, nebulizer or liquid doseinstillation (LDI) technique to provide for glycopeptide delivery. Withrespect to inhalation therapies, those skilled in the art willappreciate that where a hollow and porous microparticle composition isemployed, the composition is particularly amenable for delivery via aDPI. Conventional DPIs comprise powdered formulations and devices wherea predetermined dose of medicament, either alone or in a blend withlactose earner particles, is delivered as an aerosol of dry powder forinhalation.

The medicament is formulated in a way such that it readily dispersesinto discrete particles with an MMD between 0.5 to 20 μm, for examplefrom 0.5-5 μm, and are further characterized by an aerosol particle sizedistribution less than about 10 μm mass median aerodynamic diameter(MMAD), and in some embodiments, less than 5.0 μm. The MMAD of thepowders will characteristically range from about 0.5-10 μm, from about0.5-5.0 μm, or from about 0.5-4.0 μm.

The powder is actuated either by inspiration or by some externaldelivery force, such as pressurized air. Examples of DPIs suitable foradministration of the particulate compositions of the present inventionare disclosed in U.S. Pat. Nos. 5,740,794, 5,785,049, 5,673,686, and4,995,385 and PCT application Nos. 00/72904, 00/21594, and 01/00263, thedisclosure of each of which is incorporated by reference in theirentireties for all purposes. DPI formulations are typically packaged insingle dose units such as those disclosed in the aforementioned patentsor they employ reservoir systems capable of metering multiple doses withmanual transfer of the dose to the device.

The compositions disclosed herein may also be administered to the nasalor pulmonary air passages of a patient via aerosolization, such as witha metered dose inhaler (MDI). Breath activated MDIs are also compatiblewith the methods provided herein.

Along with the aforementioned embodiments, the compositions disclosedherein may be delivered to a patient in need thereof via a nebulizer,e.g., a nebulizer disclosed in PCT WO 99/16420, the disclosure of whichis hereby incorporated in its entirety by reference, in order to providean aerosolized medicament that may be administered to the pulmonary airpassages of the patient. A nebulizer type inhalation delivery device cancontain the compositions of the present invention as a solution, usuallyaqueous, or a suspension. For example, the prostacyclin compound orcomposition can be suspended in saline and loaded into the inhalationdelivery device. In generating the nebulized spray of the compositionsfor inhalation, the nebulizer delivery device may be drivenultrasonically, by compressed air, by other gases, electronically ormechanically (e.g., vibrating mesh or aperture plate). Vibrating meshnebulizers generate fine particle, low velocity aerosol, and nebulizetherapeutic solutions and suspensions at a faster rate than conventionaljet or ultrasonic nebulizers. Accordingly, the duration of treatment canbe shortened with a vibrating mesh nebulizer, as compared to a jet orultrasonic nebulizer. Vibrating mesh nebulizers amenable for use withthe methods described herein include the Philips Respironics I-Neb®, theOmron MicroAir, the Nektar Aeroneb®, and the Pari eFlow®.

The nebulizer may be portable and hand held in design, and may beequipped with a self contained electrical unit. The nebulizer device maycomprise a nozzle that has two coincident outlet channels of definedaperture size through which the liquid formulation can be accelerated.This results in impaction of the two streams and atomization of theformulation. The nebulizer may use a mechanical actuator to force theliquid formulation through a multiorifice nozzle of defined aperturesize(s) to produce an aerosol of the formulation for inhalation. In thedesign of single dose nebulizers, blister packs containing single dosesof the formulation may be employed.

In the present invention, the nebulizer may be employed to ensure thesizing of particles is optimal for positioning of the particle within,for example, the pulmonary membrane.

Upon nebulization, the nebulized composition (also referred to as“aerosolized composition”) is in the form of aerosolized particles. Theaerosolized composition can be characterized by the particle size of theaerosol, for example, by measuring the “mass median aerodynamicdiameter” or “fine particle fraction” associated with the aerosolizedcomposition. “Mass median aerodynamic diameter” or “MMAD” is normalizedregarding the aerodynamic separation of aqua aerosol droplets and isdetermined by impactor measurements, e.g., the Andersen Cascade Impactor(ACI) or the Next Generation Impactor (NGI). The gas flow rate, in oneembodiment, is 2example8 Liter per minute for the ACI and 15 liters perminute for the NGI.

“Geometric standard deviation” or “GSD” is a measure of the spread of anaerodynamic particle size distribution. Low GSDs characterize a narrowdroplet size distribution (homogeneously sized droplets), which isadvantageous for targeting aerosol to the respiratory system. Theaverage droplet size of the nebulized composition provided herein, inone embodiment is less than 5 μm or about 1 μm to about 5 μm, and has aGSD in a range of 1.0 to 2.2, or about 1.0 to about 2.2, or 1.5 to 2.2,or about 1.5 to about 2.2.

“Fine particle fraction” or “FPF,” as used herein, refers to thefraction of the aerosol having a particle size less than 5 μm indiameter, as measured by cascade impaction. FPF is usually expressed asa percentage.

In one embodiment, the mass median aerodynamic diameter (MMAD) of thenebulized composition is about 1 μm to about 5 μm, or about 1 μm toabout 4 μm, or about 1 μm to about 3 μm or about 1 μm to about 2 μm, asmeasured by the Anderson Cascade Impactor (ACI) or Next GenerationImpactor (NGI). In another embodiment, the MMAD of the nebulizedcomposition is about 5 μm or less, about 4 μm or less, about 3 μm orless, about 2 μm or less, or about 1 μm or less, as measured by cascadeimpaction, for example, by the ACI or NGI.

In one embodiment, the MMAD of the aerosol of the pharmaceuticalcomposition is less than about 4.9 μm, less than about 4.5 μm, less thanabout 4.3 μm, less than about 4.2 μm, less than about 4.1 μm, less thanabout 4.0 μm or less than about 3.5 μm, as measured by cascadeimpaction.

In one embodiment, the MMAD of the aerosol of the pharmaceuticalcomposition is about 1.0 μm to about 5.0 μm, about 2.0 μm to about 4.5μm, about 2.5 μm to about 4.0 μm, about 3.0 μm to about 4.0 μm or about3.5 μm to about 4.5 μm, as measured by cascade impaction (e.g., by theACI or NGI).

In one embodiment, the FPF of the aerosolized composition is greaterthan or equal to about 50%, as measured by the ACI or NGI, greater thanor equal to about 60%, as measured by the ACI or NGI or greater than orequal to about 70%, as measured by the ACI or NGI. In anotherembodiment, the FPF of the aerosolized composition is about 50% to about80%, or about 50% to about 70% or about 50% to about 60%, as measured bythe NGI or ACI.

In one embodiment, a metered dose inhalator (MDI) is employed as theinhalation delivery device for the compositions of the presentinvention. In a further embodiment, the prostacyclin compound issuspended in a propellant (e.g., hydrofluorocarbon) prior to loadinginto the MDI. The basic structure of the MDI comprises a metering valve,an actuator and a container. A propellant is used to discharge theformulation from the device. The composition may consist of particles ofa defined size suspended in the pressurized propellant(s) liquid, or thecomposition can be in a solution or suspension of pressurized liquidpropellant(s). The propellants used are primarily atmospheric friendlyhydrofluorocarbons (HFCs) such as 134a and 227. The device of theinhalation system may deliver a single dose via, e.g., a blister pack,or it may be multi dose in design. The pressurized metered doseinhalator of the inhalation system can be breath actuated to deliver anaccurate dose of the lipid-containing formulation. To insure accuracy ofdosing, the delivery of the formulation may be programmed via amicroprocessor to occur at a certain point in the inhalation cycle. TheMDI may be portable and hand held.

In one embodiment, a dry powder inhaler (DPI) is employed as theinhalation delivery device for the compositions of the presentinvention.

In one embodiment, the DPI generates particles having an MMAD of fromabout 1 μm to about 10 μm, or about 1 μm to about 9 μm, or about 1 μm toabout 8 μm, or about 1 μm to about 7 μm, or about 1 μm to about 6 μm, orabout 1 μm to about 5 μm, or about 1 μm to about 4 μm, or about 1 μm toabout 3 μm, or about 1 μm to about 2 μm in diameter, as measured by theNGI or ACI. In another embodiment, the DPI generates particles having anMMAD of from about 1 μm to about 10 μm, or about 2 μm to about 10 μm, orabout 3 μm to about 10 μm, or about 4 μm to about 10 μm, or about 5 μmto about 10 μm, or about 6 μm to about 10 μm, or about 7 μm to about 10μm, or about 8 μm to about 10 μm, or about 9 μm to about 10 μm, asmeasured by the NGI or ACI.

In one embodiment, the MMAD of the particles generated by the DPI isabout 1 μm or less, about 9 μm or less, about 8 μm or less, about 7 μmor less, 6 μm or less, 5 μm or less, about 4 μm or less, about 3 μm orless, about 2 μm or less, or about 1 μm or less, as measured by the NGIor ACI.

In one embodiment, each administration comprises 1 to 5 doses (puffs)from a DPI, for example, 1 dose (1 puff), 2 dose (2 puffs), 3 doses (3puffs), 4 doses (4 puffs) or 5 doses (5 puffs). The DPI, in oneembodiment, is small and transportable by the patient.

In one embodiment, the MMAD of the particles generated by the DPI isless than about 9.9 μm, less than about 9.5 μm, less than about 9.3 μm,less than about 9.2 μm, less than about 9.1 μm, less than about 9.0 μm,less than about 8.5 μm, less than about 8.3 μm, less than about 8.2 μm,less than about 8.1 μm, less than about 8.0 μm, less than about 7.5 μm,less than about 7.3 μm, less than about 7.2 μm, less than about 7.1 μm,less than about 7.0 μm, less than about 6.5 μm, less than about 6.3 μm,less than about 6.2 μm, less than about 6.1 μm, less than about 6.0 μm,less than about 5.5 μm, less than about 5.3 μm, less than about 5.2 μm,less than about 5.1 μm, less than about 5.0 μm, less than about 4.5 μm,less than about 4.3 μm, less than about 4.2 μm, less than about 4.1 μm,less than about 4.0 μm or less than about 3.5 μm, as measured by the NGIor ACI.

In one embodiment, the MMAD of the particles generated by the DPI isabout 1.0 μm to about 10.0 μm, about 2.0 μm to about 9.5 μm, about 2.5μm to about 9.0 μm, about 3.0 μm to about 9.0 μm, about 3.5 μm to about8.5 μm or about 4.0 μm to about 8.0 μm.

In one embodiment, the FPF of the prostacyclin particulate compositiongenerated by the DPI is greater than or equal to about 40%, as measuredby the ACI or NGI, greater than or equal to about 50%, as measured bythe ACI or NGI, greater than or equal to about 60%, as measured by theACI or NGI, or greater than or equal to about 70%, as measured by theACI or NGI. In another embodiment, the FPF of the aerosolizedcomposition is about 40% to about 70%, or about 50% to about 70% orabout 40% to about 60%, as measured by the NGI or ACI.

EXAMPLES

The present invention is further illustrated by reference to thefollowing Examples. However, it is noted that these Examples, like theembodiments described above, are illustrative and are not to beconstrued as restricting the scope of the invention in any way.

Example 1—Synthesis of LGPC Derivatives

Lipo glycopeptide cleavable (LGPC) derivatives were prepared as follows.

Reductive Amination

To a reactor vessel equipped with temperature control and agitation wasadded anhydrous DMF and DIPEA. The resulting solution was heated to 65°C. with agitation and vancomycin HCl was added slowly in portions.Heating was continued until all of vancomycin HCl had dissolved (5-10min).

The beige colored solution was allowed to cool to room temperature afterwhich a solution of the desired aldehyde dissolved in DMF was added over5-10 min. The resulting solution was allowed to stir overnight,typically producing a clear red-yellow solution. MeOH and TFA wereintroduced and stirring was further continued for at least 2 h. At theend of the stirring period, the imine forming reaction mixture wasanalyzed by HPLC which was characteristically typical. Boranetert-butylamine complex was added in portions and the reaction mixturewas stirred at ambient temperature for an additional 2 h after which anin-process HPLC analysis of the reaction mixture indicated a nearquantitative reduction of the intermediate imine group. After thereaction was over, the reaction mixture was purified using reverse phaseC18 column chromatography (Phenomenex Luna 10 uM PREP C18(2) 250×21.2 mmcolumn) using gradients of water and acetonitrile, each containing 0.1%(v/v) of TFA. Fractions were evaluated using HPLC and then pertinentfractions containing the target product were pooled together for theisolation of the product via lyophilization. Typical products wereisolated as fluffy white solids. The procedure is shown at FIG. 1.

Aldehyde Preparation

Aldehydes used in the reductive amination reaction to form the LGPC canbe prepared as set forth below and in Scheme 2.

To a reaction equipped with a stir bar was added an alcohol reagentcontaining an ester or amide bond and a suitable organic solvent(typically DCM or THF). The reaction mixture was stirred forapproximately 5 min. to fully dissolve the starting material, at whichpoint sodium bicarbonate and dess-martin periodinane were added to thereaction mixture. The reaction mixture was allowed to stir for 2 hoursat which point TLC analysis was used to assess progress. In the instancethat a large amount of unreacted starting material was present, anadditional aliquot of dess-martin periodinane was added to the reactionmixture and progress was re-assessed after an additional 2 h ofstirring. Once the reaction was complete, the reaction mixture wastreated with DCM and a solution of 10% sodium thiosulfate saturated withNaHCO₃ for 90 min. The reaction mixture was then extracted with thesodium thiosulfate solutions (3×100 mL) and brine (2×100 mL) whileretaining the organic layer. The organic layer was dried over Na₂SO₄,filtered, and solvent was removed under reduced pressure to yield thetarget aldehyde. The final material was typically used without furtherpurification. However, in some instances, the aldehyde may be purifiedby either silica gel flash column chromatography or preparatory HPLC.

Cleavable Bond Formation (Ester and Amide Coupling Reactions)

Depending on the type of LGPC desired, one of the following couplingreactions is chosen to make the alcohol reactant for the aldehydesynthesis reaction.

Glycol+Acid chloride (Scheme 1). To a reaction vessel was added theappropriate glycol such as ethylene glycol and a suitable organicsolvent such as THF or DCM. Temperature was adjusted to be 0° C. andstirring was initiated. Once the temperature stabilized, triethylaminewas added in a single aliquot. Separately, a solution of the appropriateacid chloride such as decanoyl chloride and suitable organic solventsuch as THF or DCM was prepared and charged into a dosing apparatus. Theacid chloride solution was added drop wise over the course of few hourswhile stirring at 0° C. The reaction mixture was warmed to 25° C. over a2 h period and the reaction mixture was allowed to stir forapproximately 18 h at which point stirring was stopped. The reactionmixture was filtered to remove a white precipitate that had formed.Solvent was removed under reduced pressure to yield a thick, colorlessoil.

The crude material was dissolved in EtOAc and washed with saturatedNaHCO₃, and brine. The organic layer was dried over Na₂SO₄, filtered,and evaporated to dryness to yield crude product, typically as a whitesolid. The crude material was purified using prep-HPLC with a CN columnand an isocratic method with 10% isopropyl alcohol as the mobile phase.Pure fractions were combined and solvent was removed to yield the targetcompound, typically as a white solid.

Glycol+Carboxylic Acid+Coupling Reagent (Scheme 2). To a clean vesselwas added a suitable organic solvent (typically N,N-Dimethylformamide),DIPEA, the appropriate carboxylic acid such as decanoic acid, ancoupling reagent such as HATU or PyBOP, and the appropriate glycol suchas ethylene glycol. The vial was vortexed for 30 seconds to helpdissolve the compounds. The reaction was allowed to shake overnight at40° C. and ˜125 rpm. Solvent was removed under reduced pressure and thecrude reaction mixture was purified using silica gel flash columnchromatography with a gradient method using hexanes, EtOAc, and IPA asthe mobile phases. Pure fractions were combined and solvent was removedto yield the target compound, typically as a white solid.

Hydroxy Alkyl Halide+Carboxylic Acid (Scheme 3). To a vial was added asuitable organic solvent such as N,N-Dimethylformamide, the appropriateacid chloride such as decanoyl chloride, and a hydroxyl alkyl halidesuch as 2-iodoethanol. The reaction mixture was then placed in anincubated shaker set at 40° C. and ˜125 rpm where it was left to shakeovernight. Solvent was removed under reduced pressure and the residuewas subjected to liquid-liquid extraction using H₂O (40 mL) and hexanes(3×75 ml). Organic layers were combined and solvent was removed underreduced pressure. The crude material was purified via silica gel flashcolumn chromatography using a gradient method with hexanes andethylacetate as the mobile phases. Fractions of interest were combinedand solvent was removed under reduced pressure to produce the targetcompound, typically as a thick oil.

Alkyl Halide+Hydroxy Acid coupling reaction (Scheme 4). To a vial wasadded a suitable organic solvent such as N,N-Dimethylformamide, anappropriate hydroxyl acid such as glycolic acid, and an alkyl halidesuch as 1-Iododecane. The reaction mixture was then placed in anincubated shaker set at 40° C. and ˜125 rpm where it was left to shakeovernight. Solvent was removed under reduced pressure and the residuewas subjected to liquid-liquid extraction using H₂O (40 mL) and hexanes(3×75 ml). Organic layers were combined and solvent was removed underreduced pressure. The crude material was purified via silica gel flashcolumn chromatography using a gradient method with hexanes andethylacetate as the mobile phases. Fractions of interest were combinedand solvent was removed under reduced pressure to produce the targetcompound, typically as a thick oil.

Amino alcohol+Acid Chloride (Scheme 5). To a reactor vessel was addedthe appropriate amino alcohol such as ethanolamine and a suitableorganic solvent such as THF or DCM. Temperature was adjusted to be 0° C.and stirring was initiated. Once the temperature stabilizedtriethylamine was added in a single aliquot. Separately, a solution ofthe appropriate acid chloride such as decanoyl chloride and suitableorganic solvent such as THF or DCM was prepared and charged into adosing apparatus. The acid chloride solution was added drop wise overthe course of few hours while stirring at 0° C. The reaction mixture waswarmed to 25° C. over a 2 h period and the reaction mixture was allowedto stir for approximately 18 h at which point stirring was stopped. Thereaction mixture was filtered to remove a white precipitate that hadformed. Solvent was removed under reduced pressure to yield a thick,colorless oil. The crude material was dissolved in EtOAc and washed with0.1M HCl, saturated NaHCO₃, and brine. The organic layer was dried overNa₂SO₄, filtered, and evaporated to dryness to yield crude product,typically as a white solid. The crude material was purified usingprep-HPLC with a CN column and an isocratic method with 10% isopropylalcohol as the mobile phase. Pure fractions were combined and solventwas removed to yield the target compound, typically as a white solid.

Amino alcohol+Carboxylic Acid+Coupling Reagent coupling reaction (Scheme6). To a clean vessel was added a suitable organic solvent such asN,N-Dimethylformamide), DIPEA, the appropriate carboxylic acid such asdecanoic acid, a coupling reagent such as HATU or PyBOP, and theappropriate amino alcohol such as ethanolamine. The vial was vortexedfor 30 seconds to help dissolve the compounds. The reaction was allowedto shake overnight at 40° C. and ˜125 rpm. Solvent was removed underreduced pressure and the crude reaction mixture was purified usingsilica gel flash column chromatography with a gradient method usinghexanes, EtOAc, and IPA as the mobile phases. Pure fractions werecombined and solvent was removed to yield the target compound, typicallyas a white solid.

Alkyl amine+Hydroxy Acid+Coupling Reagent coupling reaction (Scheme 7).To a clean vessel was added a suitable organic solvent such asN,N-Dimethylformamide), DIPEA, the appropriate hydroxy acid such asglycolic acid, a coupling reagent such as HATU or PyBOP, and theappropriate alkyl amine such 1-aminodecane. The vial was vortexed for 30s to help dissolve the compounds. The reaction was allowed to shakeovernight at 40° C. and ˜125 rpm. Solvent was removed under reducedpressure and the crude reaction mixture was purified using silica gelflash column chromatography with a gradient method using hexanes, EtOAc,and IPA as the mobile phases. Pure fractions were combined and solventwas removed to yield the target compound, typically as a white solid.

Example 2—Synthesis of LGPC Derivative RV65

Ester Bond Coupling (Scheme 8).

To a clean 20 mL scintillation vial was added N,N-Dimethylformamide (5mL, Potassium Carbonate (0.862 g, 6.24 mmol), Laurie acid (0.5 g, 2.5mmol), and 2-iodo-ethanol (0.43 g, 0.20 mL, 2.5 mmol). The reactionmixture was then placed in an incubated shaker set at 40° C. and −125rpm where it was left to shake overnight. Solvent was removed underreduced pressure and the residue was subjected to liquid-liquidextraction using H₂O (40 mL) and hexanes (3×75 ml). Organic layers werecombined and solvent was removed under reduced pressure. The crudematerial was purified via silica gel flash column chromatography using agradient method with hexanes and ethyl acetate as the mobile phases.Fractions of interest were combined and solvent was removed underreduced pressure to produce the target compound (91.9 mg, 0.38 mmol) asa thick, slightly yellow-tinged oil.

Oxidation to Aldehyde

To a 20 mL scintillation vial was added 2-hydroxyethyl dodecanoate(0.184 g, 0.753 mmol), dess-martin periodinane (0.639 g, 1.506 mmol),and (SI) Dichloromethane (3.68 mL). The mixture was allowed to stirovernight and reaction progress was monitored via TLC. To the reactionmixture was added 2 mL of sodium thiosulfate (10% in water) and 2 mL ofsaturated sodium bicarbonate at the same time; at which point a whiteprecipitate formed, the solution turned pink, and a small amount ofbubbles were formed. The aqueous layer was washed with DCM (3×25 mL) atwhich point organic layers were combined, washed with brined, dried overNa₂SO₄, and filtered. The crude sample was evaporated to dryness underreduced pressure to produce 2-oxoethyl dodecanoate (0.26 g, 1.08 mmol)as a slightly pink-tinged solid. The final material was analyzed by TLCusing a 2,4-DNP stain to reveal the presence of an aldehyde.

Reductive Amination

To a 40 mL vial equipped a stir bar was added anhydrous DMF (20 mL) andDIPEA (0.24 mL). The resulting solution was heated to 65° C. on anincubated shaker and vancomycin HCl (1.0 g, 0.7 mmol) was added slowlyin portions. Heating was continued until all of vancomycin HCl haddissolved (5-10 min). The beige colored solution was allowed to cool toroom temperature after which a solution of 2-oxoethyl dodecanoate (250mg, 1.03 mmol) and DMF (5 mL) was added over 5-10 min. The resultingsolution was allowed to stir overnight to give a clear red-yellowsolution. MeOH (10 mL) and TFA (0.21 mL, 2.8 mmol) were introduced tothe reaction mixture producing a small amount of white smoke; thereaction mixture also turned yellow. Stirring was further continued forat least 2 h. At the end of the stirring period, the imine formingreaction mixture was analyzed by HPLC which was characteristicallytypical. Borane tert-butylamine complex (60 mg, 0.7 mmol) was added inportions and the reaction mixture was stirred at ambient temperature foran additional 2 h after which an in-process HPLC analysis of thereaction mixture indicated a near quantitative reduction of theintermediate imine group. After the reaction was over the reactionmixture is purified using reverse phase C18 column chromatography(Phenomenex Luna 10 uM PREP C18(2) 250×21.2 mm column) using gradientsof water and acetonitrile, each containing 0.1% (v/v) of TFA. Fractionswere evaluated using HPLC and then pertinent fractions containing RV65were pooled together for the isolation of the product vialyophilization. The target compound, RV65 (150 mg, 0.09 mmol, 13%overall yield), was obtained as a white solid in >97% purity (by HPLC).

Example 3—Synthesis of LGPC Derivative RV62

Coupling (Scheme 11).

To a 400 mL reactor vessel equipped with pH monitoring, stirring,temperature control, inert gas, and a dosing apparatus was set up. Tothe reactor was added ethanolamine (3.461 g, 3.42 mL, 56.66 mmol, 2.1equiv.) and THF (150 mL, 0.18 M, 25.412 Vols). The temperature wasadjusted to be 0° C., stirring was initiated at 500 rpm, and pHmonitoring was initiated. Once the temperature stabilized Triethylamine(4.095 g, 5.641 mL, 40.472 mmol, 1.5 equiv.) was added in a singlealiquot. Separately, a solution of dodecanoyl chloride (5.903 g, 6.423mL, 26.981 mmol, 1 equiv.) and THF (50 mL, 0.54 M, 8.471 Vols) wasprepared and used to fill the dosing apparatus. The dodecanoyl chloridesolution was added drop wise over the course of 5 h while controllingthe temperature at 0° C. and the pH to basic conditions. The reactionmixture temperature was warmed to 25° C. over a 2 h period and thereaction mixture was allowed to stir for approximately 18 h at whichpoint stirring was stopped. The reaction mixture was filtered to removea white precipitate that had formed. Solvent was removed under reducedpressure to yield a thick, colorless oil. The crude material wasdissolved in EtOAc (300 mL) and was washed with 0.1M HCl (3×100 mL),saturated NaHCO₃ (3×100 mL), and brine (3×100 mL). The organic layer wasdried over Na₂SO₄, filtered, and evaporated to dryness to yield 4.45 gof crude product as a white solid. The crude material was purified usingprep-HPLC with a CN column and an isocratic method with 10% isopropylalcohol as the mobile phase. Pure fractions were combined and solventwas removed to yield the target compound as a white solid (3.15 g, 12.94mmol, 48% yield).

Oxidation to Aldehyde

To a 40 mL vial equipped with a stir bar was addedN-(2-hydroxyethyl)decanamide (1 g, 4.109 mmol, 1 equiv.),dichloromethane (20 mL, 0.205 M, 20 Vols), and THF (10 mL, 0.411 M, 10Vols). The reaction mixture was stirred for approximately 5 min. tofully dissolve the starting material at which point NaHCO₃ (0.69 g,8.217 mmol, 2 equiv.) and dess-martin periodinane (2.178 g, 5.136 mmol,1.25 equiv.) were added to the reaction mixture. The reaction mixturewas allowed to stir for 2 h at which point TLC analysis indicated thereaction had reached completion. The reaction mixture was then treatedwith and a solution of 10% sodium thiosulfate saturated with NaHCO₃ for90 min. The reaction mixture was then extracted with the sodiumthiosulfate solutions (3×100 mL) and brine (2×100 mL) while retainingthe organic layer. The organic layer (DCM) was dried over Na₂SO₄,filtered, and solvent was removed under reduced pressure to yield 673.1mg (2.79 mmol, 68.9% yield) of the target compound a white solid thatwas used without further purification.

Reductive Amination

To a 400 mL reactor vessel equipped with pH monitoring, overheadstirring, temperature control, inert gas, and a dosing apparatus wasprepared. To the reactor was added anhydrous DMF (50 mL) and DIPEA(0.694 mL). The resulting solution was heated to 65° C. with stirringand vancomycin HCl (2.9 g, 2.0 mmol) was added slowly in portions.Heating was continued until all of vancomycin HCl had dissolved (5-10min). The beige colored solution was allowed to cool to 30° C. afterwhich a solution of N-(2-oxoethyl)dodecanamide (673 mg, 2.8 mmol) andDMF was added over 5-10 min. The resulting solution was allowed to stirovernight to give a clear red-yellow solution. MeOH (25 mL) and TFA(0.61 mL, 8 mmol) were introduced and stirring was further continued forat least 2 h. At the end of the stirring period, the imine formingreaction mixture was analyzed by HPLC which was characteristicallytypical. Borane tert-butylamine complex (173 mg, 2.0 mmol) was added inportions and the reaction mixture was stirred at ambient temperature foran additional 2 h after which an in-process HPLC analysis of thereaction mixture indicated a near quantitative reduction of theintermediate imine group. After the reaction was over the reactionmixture is purified using reverse phase C18 column chromatography(Phenomenex Luna 10 uM PREP C18(2) 250×21.2 mm column) using gradientsof water and acetonitrile, each containing 0.1% (v/v) of TFA. Fractionswere evaluated using HPLC and then pertinent fractions containing RV62were pooled together for the isolation of the product vialyophilization. The target compound, RV62 (600 mg, 0.35 mmol, 18%overall yield), was obtained as a white solid in >97% purity (by HPLC).

Example 4—Synthesis of LGPC Chloroeremomycin Derivative

To a 20 mL scintillation vial equipped with a stir bar was addedchloroeremomycin and a solution of copper (II) acetate in MeOH. Thereaction mixture was stirred at room temperature until thechloroeremomycin had dissolved. To the reaction mixture was then addedthe appropriate aldehyde and sodium cyanoborohydride as a 1M solution inTHF. The reaction mixture was transferred to an incubated shaker set to45° C. and reaction progress was monitored by HPLC. In some instances,it was necessary to add an additional aliquot of aldehyde reagent. Thereaction mixture was allowed to shake overnight at 45° C. The reactionmixture was cooled to RT and sodium borohydride was added to convertresidual aldehyde reagent to the corresponding alcohol. The pH wasadjusted to between 7-8 using either acetic acid or 0.1M NaOH andvolatile solvents were removed by blowing N₂(g) with gentle heat. To thereaction mixture was added acetonitrile to precipitate the crude productas an off-white solid. The reaction mixture was centrifuged and theliquid was decanted. The solid was dissolved in 10% MeCN/H₂O containing0.1% phosphoric acid to decomplex the copper at which point the solutionbriefly turned purple and then took on a yellow tinge. Preparatory HPLCwas used to purify final product and LCMS was used to confirm compoundidentity and purity.

A diagram of the reaction is provided below as scheme 14.

Example 5—C-Terminus Modification of LGPC Derivative

To a round bottom flask equipped with a stir bar was added a LPGCderivative, a 1:1 solution of DMF:DMSO, and DIPEA. To the reactionmixture was then added HBTU and the appropriate amine (e.g.,3-(dimethylamino)-1-propylamine). Reaction progress was monitored byHPLC. Once complete, the reaction was quenched upon addition of 1:1H₂O:MeOH. The crude material was then purified using reverse phase C18preparatory HPLC. Purified fractions were lyophilized to yield thetarget products, typically as a white fluffy powder in modest yield andhigh purity.

Example 6—Resorcinol-Like Modification of LGPC Derivative

To a round bottom flask equipped with a stir bar was added(Aminomethyl)phosphoric acid, water, and DIPEA. The reaction mixture wasallowed to stir for 15 minutes at room temperature. To the reactionmixture was then added acetonitrile and formaldehyde, 37% solution inH₂O. The reaction mixture was allowed to stir for an additional 15 min.at which point a LGPC derivative and additional DIPEA were added.Reaction progress was closely monitored using HPLC. Once complete thereaction mixture was purified using reverse phase C18 preparative HPLC.Purified fractions were lyophilized to yield the target product as awhite fluffy powder.

Example 7—Minimum Inhibitory Concentration (MIC) of Compounds of Formula(II)

Compounds of the invention were evaluated for their ability to inhibitbacterial growth in two MRS A strains—MRS A 1556 and MRS A 29213. Theminimal inhibitory concentrations MICs are summarized in Table 1. Table2 provides MIC concentrations for metabolites of the ester and amidecompounds, RV80 (metabolite of RV65 ester) and RV82 (metabolite of RV62amide). MIC values for vancomycin and telavancin are also provided.

MIC Testing: Glycopeptide compounds were dissolved in 100% DMSO. Invitro activities were determined using CLSI-guided broth susceptibilitytesting to measure drug minimum inhibitory concentrations (MICs) of thecompounds against the quality control strain ATCC 29213 (MSSA) and theMRS A isolate ATCC BAA-1556.

TABLE 1 MIC Values, μg/mL MRSA MSSA Compound Class 1556 29213 R¹ R² R³R⁴ RV90 Ester 0.250 0.250 —(CH₂)₂—O—C(O)— OH H H (CH₂)₆—CH₃ RV67 Ester0.157 0.094 —(CH₂)₂—O—C(O)— OH H H (CH₂)₇—CH₃ RV54 Ester 0.167 0.146—(CH₂)₂—O—C(O)— OH H H (CH₂)₈—CH₃ RV66 Ester 0.125 0.125 —(CH₂)₂—O—C(O)—OH H H (CH₂)₉—CH₃ RV65 Ester 0.094 0.063 —(CH₂)₂—O—C(O)— OH H H(CH₂)₁₀—CH₃ RV88 Ester 0.125 0.125 —(CH₂)₂—O—C(O)— OH H H (CH₂)₁₂—CH₃RV89 Ester 1.000 0.500 —(CH₂)₂—O—C(O)— OH H H (CH₂)₁₄—CH₃ RV55 Ester0.250 0.250 —(CH₂)₃—O—C(O)— OH H H (CH₂)₉—CH₃ RV93 Amide 0.125 0.125—(CH₂)₂—NH—C(O)— OH H H (CH₂)6—CH₃ RV60 Amide 0.063 0.063—(CH₂)₂—NH—C(O)— OH H H (CH₂)₇—CH₃ RV56 Amide 0.063 0.063—(CH₂)₂—NH—C(O)— OH H H (CH₂)₈—CH₃ RV61 Amide 0.031 0.031—(CH₂)₂—NH—C(O)— OH H H (CH₂)₉—CH₃ RV62 Amide 0.023 0.023—(CH₂)₂—NH—C(O)— OH H H (CH₂)₁₀—CH₃ RV92 Amide 0.031 0.023—(CH₂)₂—NH—C(O)— OH H H (CH₂)₂—CH₃ RV91 Amide 0.250 0.188—(CH₂)₂—NH—C(O)— OH H H (CH₂)₁₄—CH₃ RV94 Amide 0.031 0.031 —CH₂—C(O)—NH—OH H H (CH₂)₉—CH₃ RV95 Amide 0.031 0.031 —CH₂—C(O)—NH— OH H H(CH₂)₁₁—CH₃ RV72 Amide 0.5 0.5 —(CH₂)₂—NH—C(O)— NH—(CH₂)₃— H H (CH₂)₉—CH₃ N(CH₃)₂ RV73 Amide 0.5 0.5 —(CH₂)₂—NH—C(O)— OH H CH₂—NH— (CH₂)₉—CH₃CH₂—PO₃H₂

TABLE 2 MIC Values, μg/mL MRSA MSSA Compound 1556 29213 Vancomycin 1 1Telavancin 0.063 0.063 RV80 1 1 RV82 3 3

MIC values for amide derivatives were lower than the ester derivatives(Table 1). RV62 was found to be about 3×more efficacious than RV65, theester with the lowest measured MIC.

Example 8—Degradation of RV62 and RV65

RV62 and RV65 degradation was determined according to the followingprocedures.

Compounds were dissolved and diluted with 1 mM Tris buffer (pH 6.99) toachieve a concentration of 54 μg/mL (stock solution). 0.5 mL of stocksolution was further diluted with acetonitrile to a total volume of 10mL. The stock solution was incubated at 40° C., with samples withdrawnat 3, 6, 24, and 72 h and tested by HPLC.

HPLC method: Samples were injected onto a 100×2.1 mm Waters CortecsHILIC with a particle size of 1.6 μm. The mobile phase consisted ofwater (0.1% formic acid) and acetonitrile (0.1% formic acid). Theanalytic method utilized a gradient from 10% water (0.1% formicacid)/90% acetonitrile (0.1% formic acid) to 70% water (0.1% formicacid)/30% acetonitrile (0.1% formic acid). The HPLC instrument wasequipped with a UV detector (280 nm). Compounds were identified by mass.

FIG. 3 shows the extent of hydrolysis of RV62 and RV65 at 3, 6, 24, and72 h. The amount of cleaved glycopeptide was found to increase steadilyup to the 24 h time point for RV65. For RV65, between 24 and 72 h, therate of cleavage appears to plateau, such that at 72 h, the peak areafor the cleaved glycopeptide was determined to be about 42% of thetotal.

Example 9—Enzyme Mediated Hydrolysis of LGPC Glycopeptide EsterDerivatives

The respective LGPC was dissolved in propanol:TBA:H2O (1:1:1) at ˜3mg/mL, with DSPE-PEG2000 (˜1.5 mg/mL), and lactosedeucine (7:3 at ˜20mg/mL). The solution was flash frozen and lyophilized. The lyophilizedcake was suspended in PBS (pH=8.0) at 2 mg/mL LGPC. The LGPC wassuspended at 0.5-1 mg/mL in PBS (pH adjusted to 8.0 with NaOH) andplaced at 37° C. in the presence and absence of esterase (0.2 U/mL).Aliquots were removed at preselected time intervals of 0, 15, 30, 45,60, 90 and 120 min. Aliquots (125 μL) were diluted in 500 μL 1:1acetonitrile (ACN):H₂O with 0.1% formic acid to stop enzymaticdegradation. Diluted samples were analyzed by HPLC to determine therelative peak area for the parent and the metabolite for each LGPCtested.

FIG. 4 is a graph of percent LGPC degradation as a function of time.Esterase mediated hydrolysis of ester LGPC derivatives is chain lengthdependent.

Example 10—Metabolism of RV62 and RV65 in Rat Plasma

RV62 and RV65 were dissolved in 100% DMSO. Stock solutions were dilutedusing rat plasma to contain less than 1% organic solvent with a finaldrug concentration of 50 μg/mL. Samples were briefly vortexed and thenincubated in a shaker set to 37° C. and 300 rpm. Aliquots were removedat specified time points and store at −80° C. until extraction andanalysis. Samples were extracted using a solution of 10% TCA andanalyzed using LCMS.

The hydrolysis of amide RV62 and ester RV65 incubated in rat plasma wasdetermined (FIG. 5). That data shows that RV65 (ester) was metabolizedfaster in plasma compared to RV62 (amide). For RV65, ˜90% of degradant(RV80) was detected in plasma after only 6 h incubation. In contrast,only 6% of degradant of RV62 (RV82) was detected in plasma even after 24h incubation. As such, under the test conditions, the ester moiety wasfound to be more labile than the corresponding amide moiety.

Example 11—Pharmacokinetics (PK) of RV62 and its Hydrolysis ProductRV82, Given by Nose Only Inhalation in Rats

The structures are RV62 and RV82 are provided below.

Male Sprague-Dawley rats from Charles River Laboratories weighingbetween 250 g and 300 g at the start of dosing were used in the study.

RV62 solution 5 mg/mL in Bicine buffer 0.8 mg/mL at pH 9.5 was preparedprior to animal dosing. RV62 was administered using Aeroneb nebulizer(Aerogen) which delivers a mass mean aerosol diameter between 2.5 to 4μm and a range of 0.2-0.4 mL/min of nebulization rate. The volume ofmaterial to be nebulized was 6 mL, and the total administration time was˜20 min.

On the day of dosing, the eleven rats were placed into the nose-conerestraint chambers which are connected to a 12-port nose-only inhalationchamber (CH Technologies). The test article was delivered from thenebulizer to the chamber with an airflow of 6 L/min. At the end of thecompound exposure, the rats were either returned to their cage orsacrificed at 0.5 h after the end of nebulization which was defined asthe immediately post dose (IPD) collection.

For the terminal time points, rats were anesthetized with 2% isofluraneinhaled with pure oxygen and blood samples of 2.0 mL were obtained byheart puncture and transferred into a 2.0 mL K2-EDTA tube. The tubeswere centrifuged at 4° C. to separate the plasma and aliquoted intothree conical tubes and stored at −50° C. Lungs were extracted, weighed,and stored at −50° C. for subsequent analysis of lung drugconcentrations. RV62 and RV82 were measured in both blood plasma and thelung by LC-MS/MS method. Results of the study are provided at FIG. 6(lung) and FIG. 7 (blood plasma).

All, documents, patents, patent applications, publications, productdescriptions, and protocols which are cited throughout this applicationare incorporated herein by reference in their entireties for allpurposes.

The embodiments illustrated and discussed in this specification areintended only to teach those skilled in the art the best way known tothe inventors to make and use the invention. Modifications and variationof the above-described embodiments of the invention are possible withoutdeparting from the invention, as appreciated by those skilled in the artin light of the above teachings. It is therefore understood that, withinthe scope of the claims and their equivalents, the invention may bepracticed otherwise than as specifically described.

The invention claimed is:
 1. A method for treating a Gram positivepulmonary bacterial infection in a patient in need thereof, the methodcomprising: administering to the lungs of the patient via a nebulizer,dry powder inhaler (DPI) or metered dose inhaler (MDI), a compositioncomprising an effective amount of a compound of Formula (II), or apharmaceutically acceptable salt thereof:

wherein, R¹ is —(CH₂)_(n1)—C(O)—NH—(CH₂)_(n2)—CH₃;—(CH₂)_(n1)—NH—C(O)—(CH₂)_(n2)—CH₃; or—(CH₂)_(n1)—O—C(O)—(CH₂)_(n2)—CH₃; n1 is 1, 2, 3, 4 or 5; n2 is 6, 7, 8,9, 10, 11, 12, 13, 14 or 15; R² is OH or NH—(CH₂)_(q)—R⁵; q is 1, 2, 3,4, or 5; R³ is H or

R⁴ is H or CH₂—NH—CH₂—PO₃H₂; and R⁵ is —N(CH₃)₂, —N⁺(CH₃)₃,—N⁺(CH₃)₂(n-C₁₄H₂₉), or


2. The method of claim 1, wherein R¹ is—(CH₂)_(n1)—NH—C(O)—(CH₂)_(n2)—CH₃ or —(CH₂)_(n1)—O—C(O)—(CH₂)_(n2)—CH₃.3. The method of claim 1, wherein R¹ is—(CH₂)_(n1)—O—C(O)—(CH₂)_(n2)—CH₃.
 4. The method of claim 1, wherein R¹is —(CH₂)_(n1)—NH—C(O)—(CH₂)_(n2)—CH₃.
 5. The method of claim 1, whereinR¹ is —(CH₂)_(n1)—C(O)—NH—(CH₂)_(n2)—CH₃.
 6. The method of claim 5,wherein n1 is
 1. 7. The method of claim 5, wherein n2 is 9, 10, 11, 12,13,
 14. 8. The method of claim 6, wherein n2 is 9, 10, 11, 12 or
 13. 9.The method of claim 1, wherein R¹ is —(CH₂)_(n1)—C(O)—NH—(CH₂)_(n2)—CH₃,R² is OH, R³ is H, R⁴ is H, n1 is 1 and n2 is
 9. 10. The method of claim1, wherein the administering is carried out via a dry powder inhaler.11. The method of claim 1, wherein the Gram-positive pulmonary bacterialinfection is a Gram-positive pulmonary cocci infection.
 12. The methodof claim 11, wherein the Gram-positive pulmonary cocci infection is aStaphylococcus pulmonary infection.
 13. The method of claim 11, whereinthe Gram-positive pulmonary cocci infection is an Enterococcus pulmonaryinfection.
 14. The method of claim 11, wherein the Gram-positivepulmonary cocci infection is a Streptococcus pulmonary infection. 15.The method of claim 12, wherein the Staphylococcus pulmonary infectionis a Staphylococcus aureus (S. aureus) pulmonary infection.
 16. Themethod of claim 15, wherein the S. aureus pulmonary infection is amethicillin-resistant S. aureus (MRSA) pulmonary infection.
 17. Themethod of claim 1, wherein the patient is a cystic fibrosis patient. 18.The method of claim 4, wherein n1 is
 2. 19. The method of claim 18,wherein n2 is
 10. 20. The method of claim 1, wherein R¹ is—(CH₂)_(n1)—NH—C(O)—(CH₂)_(n2)—CH₃, R² is OH, R³ is H, R⁴ is H, n1 is 2and n2 is 10.